Lipid construct for delivery of insulin to a mammal

ABSTRACT

The instant invention is drawn to a hepatocyte targeted composition comprising insulin associated with a lipid construct comprising an amphipathic lipid and an extended amphipathic lipid that targets the construct to a receptor displayed by an hepatocyte. The composition can comprise a mixture of free insulin and insulin associated with the complex. The composition can be modified to protect insulin and the complex from degradation. The invention also includes methods for the manufacture of the composition and loading insulin into the composition and recycling various components of the composition. Methods of treating individuals inflicted with diabetes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/916,115, filed Jun. 12, 2013, now allowed, which is a continuationof, and claims priority to, U.S. patent application Ser. No. 11/920,905,filed Nov. 18, 2009, which is a U.S. national phase application filedunder 35 U.S.C. § 371 claiming benefit to International PatentApplication No. PCT/US2006/019119, filed May 16, 2006, which: is acontinuation-in-part of, and claims priority to, U.S. patent applicationSer. No. 11/384,728, filed Mar. 20, 2006, now issued as U.S. Pat. No.7,871,641, is a continuation-in-part of, and claims priority to, U.S.patent application Ser. No. 11/384,659, filed Mar. 20, 2006, now issuedas U.S. Pat. No. 7,858,116; and claims the benefit pursuant to 35 U.S.C.§ 119(e) of U.S. Provisional Application No. 60/683,878, filed May 23,2005, all of which applications are hereby incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

Diabetes is a disorder affecting large numbers of people worldwide.Management approaches to control Type I and Type II diabetes aimprimarily at normalizing blood glucose levels to prevent short- andlong-term complications. Many patients require multiple daily injectionsof an insulin to control their diabetes. Several insulin products havebeen produced that control blood sugar levels over differing timeintervals. Several products combine various forms of insulin in anattempt to provide a preparation which controls glucose levels over awider period of time.

Previous attempts to normalize blood glucose levels in Type I and TypeII diabetic patients have centered on the subcutaneous administration ofinsulin in various time-released formulations, such as ultralente andhumulin NPH insulin pharmaceutical products. These formulations haveattempted to delay and subsequently control the bio-distribution ofinsulin by regulating release of insulin to peripheral tissues with theexpectation that sustained management of insulin bio-availability willlead to better glucose control. Glargine insulin is a long-acting formof insulin in which insulin is released from the subcutaneous tissuearound the site of injection into the bloodstream at a relativelyconstant rate throughout the day. Although glargine insulin is releasedat a constant rate throughout the day, the released insulin reaches awide range of systems within the body rather than being delivered totargeted areas of the body. What is needed is a composition of insulinwhere a portion of the dosed insulin is released at a relativelyconstant rate throughout the day and another portion of insulin that istime released from the site of administration and targeted for deliveryto the liver to better control glucose production.

There is, therefore, an unmet need in the art for compositions andmethods of managing blood glucose levels in Type I and Type II diabeticpatients. The present invention meets these needs by providing along-acting composition comprising insulin that is free and insulin thatis associated with a lipid construct targeted for delivery tohepatocytes. A lipid construct is a lipid/phospholipid particle in whichindividual lipid molecules cooperatively interact to create a bipolarlipid membrane which encloses and isolates a portion of the medium inwhich it was formed. The lipid construct releases free insulin over timeas well as targets a portion of the remaining insulin to the hepatocytesin the liver to better control glucose storage and production.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention includes a lipid constructcomprising an amphipathic lipid and an extended amphipathic lipid,wherein the extended amphipathic lipid comprises proximal, medial anddistal moieties, wherein the proximal moiety connects the extendedamphipathic lipid to the construct, the distal moiety targets theconstruct to a receptor displayed by a hepatocyte, and the medial moietyconnects the proximal and distal moieties.

In another aspect, the lipid construct further comprises at least oneinsulin.

In still another aspect, the at least one insulin is selected from thegroup consisting of insulin lispro, insulin aspart, regular insulin,insulin glargine, insulin zinc, human insulin zinc extended, isophaneinsulin, human buffered regular insulin, insulin glulisine, recombinanthuman regular insulin, recombinant human insulin isophane, premixedcombinations of any of the aforementioned insulins, a derivativethereof, and a combination of any of the aforementioned insulins.

In another aspect, the lipid construct further comprises an insolubleform of at least one insulin associated with the lipid construct.

In yet another aspect, the amphipathic lipid comprises at least onelipids selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetylphosphate, 1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycero)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),derivatives thereof, and mixtures of any of the foregoing compounds.

In one aspect, the proximal moiety of the extended amphipathic lipidcomprises at least one, but not more than two, long acyl hydrocarbonchains bound to a backbone, wherein each hydrocarbon chain isindependently selected from the group consisting of a saturatedhydrocarbon chain and an unsaturated hydrocarbon chain.

In another aspect, the backbone comprises glycerol.

In still another aspect, the distal moiety of the extended amphipathiclipid comprises at least one member selected from the group consistingof biotin, a biotin derivative, iminobiotin, an iminobiotin derivative,biocytin, a biocytin derivative, iminobiocytin, an iminobiocytinderivative and a hepatocyte specific molecule that binds to a receptorin a hepatocyte.

In yet another aspect, the extended amphipathic lipid is selected fromthe group consisting of N-hydroxysuccinimide (NHS) biotin;sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin;sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin;sulfo-N-hydroxysuccinimide-S—S-biotin; biotin-BMCC; biotin-HPDP;iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytinhydrazide; biotin cadaverine; carboxybiotin; photobiotin; ρ-aminobenzoylbiocytin trifluoroacetate; ρ-diazobenzoyl biocytin; biotin DHPE;biotin-X-DHPE; 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; α-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol)amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; biotin6-O-phospho-α-D-mannopyranoside; andpolychromium-poly(bis)-N-[2,6-(diisopropylphenyl) carbamoyl methylimino]diacetic acid.

In one aspect, the medial moiety of the extended amphipathic lipidcomprises a thio-acetyl triglycine polymer or a derivative thereof,wherein the extended amphipathic lipid molecule extends outward from thesurface of the lipid construct.

In another aspect, the lipid construct further comprises at least oneinsulin associated with a water insoluble target molecule complex,wherein the complex comprises a plurality of linked individual units,the individual units comprise: a bridging component selected from thegroup consisting of a transition element, an inner transition element, aneighbor element of the transition element and a mixture of any of theforegoing elements, and a complexing component, provided that when thetransition element is chromium, a chromium target molecule complex isformed.

In yet another aspect, the lipid construct further comprises at leastone insulin that is not associated with the target molecule complex.

In a further aspect, the bridging component is chromium.

In one aspect, the complexing component comprisespoly(bis)-[(N-(2,6-diisopropylphenyl)carbamoyl methyl) iminodiaceticacid].

In another aspect, the distal component of the extended amphipathiclipid comprises a non-polar derivatized benzene ring or a heterobicyclicring structure.

In still another aspect, the construct comprises a positive charge, anegative charge or combinations thereof.

In one aspect, the extended amphipathic lipid comprises at least onecarbonyl moiety positioned at a distance about 13.5 angstroms or lessfrom the terminal end of the distal moiety.

In another aspect, the extended amphipathic lipid comprises at least onecarbamoyl moiety comprising a secondary amine.

In yet another aspect, the extended amphipathic lipid comprises chargedchromium in the medial position.

In a further aspect, the lipid construct further comprises celluloseacetate hydrogen phthalate.

In yet another aspect, the lipid construct further comprises at leastone charged organic molecule bound to the insulin.

In one aspect, the charged organic molecule is selected from the groupconsisting of protamines, derivatives of polylysine, highly basic aminoacid polymers, poly (arg-pro-thr)n in a mole ratio of 1:1:1, poly(DL-Ala-poly-L-lys)n in a mole ratio of 6:1, histones, sugar polymersthat contain a positive charge contributed by a primary amino group,polynucleotides with primary amino groups, carboxylated polymers andpolymeric amino acids, fragments of proteins that contain large amountsof amino acid residues with carboxyl (COO—) or sulfhydral (S—)functional groups, derivative of proteins with negatively chargedterminal acidic carboxyl groups, acidic polymers, sugar polymerscontaining negatively charged carboxyl groups, derivative thereof andcombinations of the aforemention compounds.

In another aspect, a method of manufacturing a lipid constructcomprising an amphipathic lipid and an extended amphipathic lipid,wherein the extended amphipathic lipid comprises proximal, medial anddistal moieties, wherein the proximal moiety connects the extendedamphipathic lipid to the construct, the distal moiety targets theconstruct to a receptor displayed by a hepatocyte, and the medial moietyconnects the proximal and distal moieties, comprises: creating a mixturecomprising the amphipathic lipid and an extended amphipathic lipid; andforming a suspension of the lipid construct in water.

In still another aspect, the method of manufacturing the lipid constructcomprising an insulin, an amphipathic lipid and an extended amphipathiclipid, wherein the extended amphipathic lipid comprises proximal, medialand distal moieties, wherein the proximal moiety connects the extendedamphipathic lipid to the construct, the distal moiety targets theconstruct to a receptor displayed by a hepatocyte, and the medial moietyconnects the proximal and distal moieties, comprises: creating a mixturecomprising the amphipathic lipid and an extended amphipathic lipid;forming a suspension of the lipid construct in water; and loading theinsulin into the lipid construct.

In another aspect, the step of loading the insulin into the lipidconstruct comprises equilibrium loading and non-equilibrium loading.

The still another aspect, the step of loading the insulin into the lipidconstruct comprises adding a solution containing free insulin to amixture of the lipid construct in water and allowing the insulin toremain in contact with the mixture until equilibrium is reached.

In yet another aspect, the method further comprises the step ofterminally loading the insulin into the lipid construct after themixture reaches equilibrium, wherein the solution containing freeinsulin is removed from the construct, further wherein the constructcontains insulin associated with the construct.

In one aspect, the method further comprises the step of removing thesolution containing free insulin from the lipid construct containinginsulin associated with the construct by a process selected from thegroup consisting of a rapid filtration procedure, centrifugation, filtercentrifugation, and chromatography using an ion-exchange resin orstreptavidin agarose affinity-resin gel having affinity for biotin,iminobiotin or derivates thereof.

In another aspect, the method further comprises the step of adding achromium complex comprising a plurality of linked individual units tothe lipid construct.

In still another aspect, the method further comprises the step of addingcellulose acetate hydrogen phthalate to the lipid construct.

In yet another aspect, the method further comprises the step ofreclaiming from the process at least one material selected from thegroup consisting of insulin, ion-exchange resin and streptavidin agaroseaffinity-gel.

In another aspect, the step of loading the insulin into the lipidconstruct comprises the step of adding at least one charged organicmolecule to the insulin before the insulin is loaded into the lipidconstruct.

In still another aspect, a method of increasing the bioavailability ofat least one insulin in a patient comprises: combining at least oneinsulin with a lipid construct, wherein the lipid construct comprises aplurality of non-covalent multi-dentate binding sites; and administeringthe construct containing insulin to the patient.

In another aspect, increasing the bioavailability further comprising thestep of modulating the isoelectric point of at least one activeingredient.

In yet another aspect, the insulin is selected from the group consistingof insulin lispro, insulin aspart, regular insulin, insulin glargine,insulin zinc, human insulin zinc extended, isophane insulin, humanbuffered regular insulin, insulin glulisine, recombinant human regularinsulin, recombinant human insulin isophane, premixed combinations ofany of the aforementioned insulins, a derivative thereof, and acombination of any of the aforementioned insulins.

In yet another aspect, the lipid construct comprises insulin,1,2-distearoyl-sn-glycero-3-phophocholine, cholesterol, dicetylphosphate, 1,2-dipalmitoyl-sn-glycero-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) orderivatives, and a hepatocyte receptor binding molecule.

In one aspect, the method further comprises the step of adding at leastone charged organic molecule to the insulin before the insulin iscombined with the lipid construct.

In another aspect, a method of forming a time-release composition thatprovides increased bio-distribution of insulin in a host comprises:removing a lipid construct from a bulk phase media by binding theconstruct through lipids comprising iminobiotin or an iminobiotinderivative to streptavidin agarose affinity-gel at pH 9.5 or greater;separating the construct from the bulk phase media; and releasing theconstruct from the affinity-gel by adjusting the pH of an aqueousmixture of the affinity gel to pH 4.5, wherein, the released constructcontains insoluble insulin; wherein upon administration of the constructto a warm-blooded host the insulin is resolubilized under thephysiological pH conditions in the host.

In still another aspect, a method of treating a patient afflicted withdiabetes comprises administering to the patient an effective amount of alipid construct comprising insulin associated with the construct.

In yet another aspect, the insulin is selected from the group consistingof insulin lispro, insulin aspart, regular insulin, insulin glargine,insulin zinc, human insulin zinc extended, isophane insulin, humanbuffered regular insulin, insulin glulisine, recombinant human regularinsulin, recombinant human insulin isophane, premixed combinations ofany of the aforementioned insulins, a derivative thereof, and acombination of any of the aforementioned insulins.

In one aspect, the lipid construct further comprises a target moleculecomplex, wherein the complex comprises a plurality of linked individualunits, further wherein the linked individual units comprises: a bridgingcomponent selected from the group comprising a transition element, aninner transition element, a neighbor element of the transition elementand a mixture of any of the foregoing elements; and a complexingcomponent; provided that when the transition element is chromium, achromium target molecule complex is formed.

In another aspect, the lipid construct further comprises insulin notassociated with the target molecule complex.

In still another aspect, the administration is oral or subcutaneous.

In yet another aspect, the insulin associated with the constructcomprises at least one charged organic molecule bound to the insulin.

In one aspect, the invention includes a method for increasing thedelivery of insulin to hepatocytes in the liver of a patient afflictedwith diabetes by administering to the patient a lipid constructcomprising insulin, an amphipathic lipid, and an extended lipid, whereinthe extended lipid comprises a moiety that binds to hepatocytereceptors, wherein the lipid construct is present in a plurality ofsizes.

In another aspect the at least one insulin is selected from the groupconsisting of insulin lispro, insulin aspart, regular insulin, insulinglargine, insulin zinc, human insulin zinc extended, isophane insulin,human buffered regular insulin, insulin glulisine, recombinant humanregular insulin, recombinant human insulin isophane, premixedcombinations of any of the aforementioned insulins, a derivativethereof, and a combination of any of the aforementioned insulins.

In still another aspect, the method further comprises protecting theinsulin within the lipid construct from hydrolytic degradation byproviding a three-dimensional structural array of lipid molecules so asto prevent access to the insulin by hydrolytic enzymes.

In yet another aspect, the method further comprises adding celluloseacetate hydrogen phthalate to the lipid construct to react withindividual lipid molecules.

In still another aspect, the method further comprises producing aninsolubilized dosage form of insulin within the lipid construct.

In one aspect, the invention includes a kit for use in treating a mammalinflicted with diabetes, the kit comprising a lipid construct, aphysiological buffer solution, an applicator, and an instructionalmaterial for the use thereof, wherein the lipid construct comprises anamphipathic lipid and an extended amphipathic lipid, wherein theextended amphipathic lipid comprises proximal, medial and distalmoieties, wherein the proximal moiety connects the extended amphipathiclipid to the construct, the distal moiety targets the construct to areceptor displayed by a hepatocyte, and the medial moiety connects theproximal and distal moieties.

In another aspect, the kit further comprises at least one insulin.

In one aspect the invention includes a hepatocyte-targeting compositioncomprises: at least one free insulin; at least one insulin associatedwith a water-insoluble target molecule complex and a lipid constructmatrix comprising at least one lipid component; wherein the targetmolecule complex is comprised of a combination of: multiple linkedindividual units, the individual units comprising: at least one bridgingcomponent selected from the group consisting of a transition element, aninner transition element, and a neighbor element of the transitionelement; and a complexing component; provided that when the transitionelement is chromium, a chromium target molecule complex is created;further wherein the target molecule complex comprises a negative charge.

In another aspect, the at least one insulin is selected from the groupconsisting of insulin lispro, insulin aspart, regular insulin, insulinglargine, insulin zinc, human insulin zinc extended, isophane insulin,human buffered regular insulin, insulin glulisine, recombinant humanregular insulin, recombinant human insulin isophane, premixedcombinations of any of the aforementioned insulins, a derivativethereof, and a combination of any of the aforementioned insulins.

In still another aspect, the insulin comprises insulin-like moieties,including fragments of insulin molecules, that have the biologicalactivity of insulins.

In yet another aspect, the lipid component comprises at least one lipidselected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine, cholesterol, cholesterololeate, dicetylphosphate, 1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dimyristoyl-sn-glycero-3-phosphate.

In one aspect, the lipid component comprises at least one lipid selectedfrom the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,cholesterol, and dicetyl phosphate.

In another aspect, the lipid component comprises a mixture of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and dicetylphosphate.

In still another aspect, the bridging component is chromium.

In yet another aspect, the complexing component comprises at least onemember selected from the group consisting of:

-   N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,6-diethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-isopropylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,3-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-tertiary butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3-butoxyphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;-   aminopyrrol iminodiacetic acid;-   N-(3-bromo-2,4,6-trimethylphenylcarbamoylmethyl) iminodiacetic acid;-   benzimidazole methyl iminodiacetic acid;-   N-(3-cyano-4,5-dimethyl-2-pyrrylcarbamoylmethyl) iminodiacetic acid;-   N-(3-cyano-4-methyl-5-benzyl-2-pyrrylcarbamoylmethyl) iminodiacetic    acid; and-   N-(3-cyano-4-methyl-2-pyrrylcarbamoylmethyl) iminodiacetic acid.

In still another aspect, the complexing component comprisespoly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid].

In one aspect, the present invention includes a method of manufacturinga hepatocyte-targeting composition comprises: creating a target moleculecomplex, wherein the complex comprises multiple linked individual unitsand a lipid construct matrix; forming a suspension of the targetmolecule complex in buffer; and combining the insulin and the targetmolecule complex.

In another aspect, a method of manufacturing a hepatocyte-targetingcomposition comprises: creating a target molecule complex, wherein thecomplex comprises multiple linked individual units and a lipid constructmatrix; forming a suspension of the target molecule complex in water;adjusting the pH of the water suspension to approximately pH 5.3;adjusting the pH of the glargine insulin to approximately 4.8; andcombining the glargine insulin and the target molecule complex, whereinthe insulin is glargine insulin.

In still another aspect, a method of manufacturing ahepatocyte-targeting composition comprises: creating a target moleculecomplex, wherein the complex comprises multiple linked individual unitsand a lipid construct matrix; forming a suspension of the targetmolecule complex in water; adjusting the pH of the water suspension toapproximately pH 5.3; adjusting the pH of the glargine insulin toapproximately 4.8; and combining the glargine insulin, the non-glargineinsulin and the target molecule complex, wherein the insulin comprisesglargine insulin and at least one non-glargine insulin.

In one aspect the present invention includes a method of treating apatient for Type I or Type II diabetes comprising administering to thepatient an effective amount of a hepatocyte-targeting composition.

In another aspect, the route of administration is selected from thegroup consisting of oral, parenteral, subcutaneous, pulmonary andbuccal.

In still another aspect, the route of administration is oral orsubcutaneous.

In one aspect the present invention includes a method of treating apatient for Type I or Type II diabetes comprising administering to thepatient an effective amount of a hepatocyte targeted composition,wherein insulin comprises glargine insulin and at least one non-glargineinsulin, further wherein the non-glargine insulin is selected from thegroup consisting of insulin lispro, insulin aspart, regular insulin,insulin glargine, insulin zinc, human insulin zinc extended, isophaneinsulin, human buffered regular insulin, insulin glulisine, recombinanthuman regular insulin, recombinant human insulin isophane, premixedcombinations of any of the aforementioned insulins, a derivativethereof, and a combination of any of the aforementioned insulins.

In another aspect, the non-glargine insulin comprises insulin-likemoieties, including fragments of insulin molecules, that have biologicalactivity of insulins.

In still another aspect, the present invention includes a method oftreating a patient for Type I or Type II diabetes comprisingadministering to the patient an effective amount of ahepatocyte-targeting composition.

In another aspect, the route of administration is selected from thegroup consisting of oral, parenteral, subcutaneous, pulmonary andbuccal.

In still another aspect, the route of administration is oral orsubcutaneous.

In yet another aspect, the present invention includes a method oftreating a patient for Type I or Type II diabetes comprisingadministering to the patient an effective amount of a hepatocytetargeted composition, wherein insulin comprises recombinant humaninsulin isophane and at least one insulin that is not recombinant humaninsulin isophane.

In another aspect, the at least one insulin that is not recombinanthuman insulin isophane comprises insulin-like moieties, includingfragments of insulin molecules, that have biological activity ofinsulins.

In one aspect, the present invention includes a kit for use in treatingType I or Type II diabetes in a mammal, the kit comprising aphysiological buffered solution, an applicator, instructional materialfor the use thereof, and a water insoluble target molecule complex,wherein the complex comprises multiple linked individual units and alipid construct matrix containing a negative charge, the multiple linkedindividual units comprising: a bridging component selected from thegroup consisting of a transition element, an inner transition element, aneighbor element of the transition element and a mixture of any of theforegoing elements, and a complexing component, provided that when thetransition element is chromium, a chromium target molecule complex iscreated, wherein the multiple linked individual units are combined withthe lipid construct matrix.

In another aspect, the kit further comprising at least one insulin,wherein the insulin is associated with the target molecule complex-,wherein the complex comprises a charge.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purposes of illustrating the invention, there are depicted inthe drawings certain embodiments of the invention. However, theinvention is not limited to the precise arrangements andinstrumentalities of the embodiments depicted in the drawings.

FIG. 1 is a depiction of an insulin binding lipid construct comprisinginsulin, amphipathic lipid molecules and an extended amphipathic lipid.

FIG. 2 is depiction of a route for manufacturing biocytin.

FIG. 3 is a depiction of a route for manufacturing iminobiocytin.

FIG. 4 is a depiction of a route for manufacturing benzoyl thioacetyltriglycine iminobiocytin (BTA-3gly-iminobiocytin).

FIG. 5 is a depiction of a route for manufacturing benzoyl thioacetyltriglycine.

FIG. 6 is a depiction of a route for manufacturing benzoyl thioacetyltriglycine sulfo-N-hydroxysiccinimide (BTA-3-gly-sulfo-NHS).

FIG. 7 is a depiction of a route for manufacturing benzoyl thioacetyltriglycine iminobiocytin (BTA-3-gly-iminobiocytin).

FIG. 8 is a depiction of a route for manufacturing a lipid anchoring andhepatocyte receptor binding molecule (LA-HRBM).

FIG. 9 is a depiction of potential sites for binding between celluloseacetate hydrogen phthalate and insulin.

FIG. 10 is a depiction of the change in structure of iminobiotin underacidic versus basic conditions.

FIG. 11 is a depiction of the chemical structure of glargine insulin.

FIG. 12 is a depiction of the chemical structure of recombinant humaninsulin isophane and a protamine protein.

FIG. 13 is a depiction of a pharmaceutical composition that combinesfree insulin and insulin associated with a water insoluble targetmolecule complex.

FIG. 14 is an outline of a method of manufacturing an insulin bindinglipid construct comprising amphipathic lipid molecules and an extendedamphipathic lipid.

FIG. 15 is an outline of the method of manufacturing a hepatocytetargeted pharmaceutical composition that combines free glargine insulinand glargine insulin associated with a water insoluble target moleculecomplex.

FIG. 16 is an outline of the method of manufacturing a hepatocytetargeted pharmaceutical composition that combines free recombinant humaninsulin isophane and recombinant human insulin isophane associated witha water insoluble target molecule complex that contains a portion ofrecombinant human regular insulin that is both free and associated witha lipid construct.

FIG. 17 indicates the concentration of glycogen present in the liver ofrats treated with various hepatocyte targeted compositions.

FIG. 18 is a graph of the concentrations of glucose in blood ofindividual patients treated once before breakfast with HDV-glargineinsulin.

FIG. 19 is a graph of the effect of a single dose of HDV-glargineinsulin on average blood glucose concentrations in patients consumingthree meals during the day.

FIG. 20 is a graph of the effect of HDV-glargine insulin on bloodglucose concentrations over time relative to blood glucoseconcentrations during fasting.

FIG. 21 is a graph of the concentrations of glucose in blood ofindividual patients treated once before breakfast with HDV-Humulin NPHinsulin.

FIG. 22 is a graph of the effect of a single dose of HDV-Humulin NPHinsulin on average blood glucose concentrations in patients consumingthree meals during the day.

FIG. 23 is a graph of the effect of HDV-Humulin NPH insulin on bloodglucose concentrations over time relative to blood glucoseconcentrations during fasting.

DETAILED DESCRIPTION OF THE INVENTION

The invention includes a hepatocyte targeted pharmaceutical compositionwhere insulin is associated with a water insoluble target moleculecomplex within the construct and the composition is targeted tohepatocytes in the liver of a patient to provide an effective means ofmanaging diabetes.

The invention includes a lipid construct comprising insulin, anamphipathic lipid and an extended amphipathic lipid (a receptor bindingmolecule). The extended amphipathic lipid comprises proximal, medial anddistal moieties. The proximal moiety connects the extended lipidmolecule to the construct, the distal moiety targets the construct to areceptor displayed by a hepatocyte, and the medial moiety connects theproximal and distal moieties.

A lipid construct is a spherical lipid and phospholipid particle inwhich individual lipid molecules cooperatively interact to create abipolar lipid membrane which encloses and isolates a portion of themedium in which it was formed. The lipid construct can target thedelivery of insulin to the hepatocytes in the liver and provide for asustained release of insulin to better control diabetes.

The invention also includes a hepatocyte targeted pharmaceuticalcomposition that combines free insulin and insulin associated with awater insoluble target molecule complex targeted to hepatocytes in theliver of a patient to provide an effective means of managing bloodglucose levels. When a mixture of different forms of insulin areassociated with a target molecule complex to create a unique mixture ofinsulin molecules, an added therapeutic benefit is achieved once theseinsulins are combined in a hepatocyte targeted lipid construct. Thecomposition of the invention can be administered by various routes,including subcutaneously or orally, for the purpose of treating mammalsafflicted with diabetes.

The invention further provides a method of manufacturing a lipidconstruct comprising insulin, an amphipathic lipid and an extendedamphipathic lipid. The extended amphipathic lipid molecule comprisesproximal, medial and distal moieties. The proximal moiety connects theextended lipid to the construct. The distal moiety targets the constructto a receptor displayed by a hepatocyte, and the medial moiety connectsthe proximal and distal moieties.

The invention also provides a method of manufacturing a compositioncomprising free insulin and insulin associated with a water insolubletarget molecule complex within the lipid construct that targets deliveryof the complex to hepatocytes. The target molecule complex comprises alipid construct matrix containing multiple linked individual units of astructure formed by a metal complex.

Additionally, the invention provides methods of treating individualsafflicted with diabetes by administering an effective dose of a lipidconstruct comprising insulin, an amphipathic lipid and an extendedamphipathic lipid, targeted for delivery to hepatocytes.

The invention also provides methods of treating individuals afflictedwith diabetes by administering an effective dose of a lipid constructcomprising insulin, an amphipathic lipid, an extended amphipathic lipidand a water insoluble target molecule complex, targeted for delivery tohepatocytes.

The invention also provides methods of treating a patient with insulinto which a polar organic compound, or mixture of compounds, is bound,thereby changing the isoelectric point of insulin. This change in theisolelectric point will change the release of insulin into the body ofpatient treated with the composition.

Additionally, the invention provides methods of managing blood glucoselevels in individuals with Type I and Type II diabetes by administeringan effective dose of a hepatocyte targeted pharmaceutical compositionthat combines free insulin and insulin associated with a water insolubletarget molecule complex targeted for delivery to hepatocytes. Thecombination of free insulin and insulin associated with a waterinsoluble target molecule complex creates a dynamic equilibrium processbetween the two forms of insulin that occurs in vivo to help control themovement of free insulin to the receptor sites of hormonal action, suchas the muscle and adipose tissue of a diabetic patient over a designatedtime period. Hepatocyte targeted insulin is also delivered to the liverof a diabetic patient over a different designated time period than freeinsulin thereby introducing new pharmacodynamic profiles of insulin whenfree insulin is released from the lipid construct. In addition, aportion of insulin that is associated with the lipid construct istargeted to the liver. This new pharmacodynamic profile of the productprovides not only long-acting basal insulin for peripheral tissues, butalso meal-time hepatic insulin stimulation for the management of hepaticglucose storage during a meal. Free insulin is released from the site ofadministration and is distributed throughout the body. Insulinassociated with a water insoluble target molecule complex is deliveredto the liver, where it is released over time from the complex. The rateof release of insulin associated with the target molecule complex isdifferent than the rate of release of free insulin from the site ofadministration. These different release rates of insulin delivery,combined with the targeted delivery of insulin associated with a lipidconstruct to the liver, provide for the normalization of glucoseconcentrations in patients with Type I and Type II diabetes. Thehepatocyte targeted composition can also comprise other types ofinsulin, or a combination of other types of insulin.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which the invention belongs. Generally, thenomenclature used herein and the laboratory procedures in organicchemistry and protein chemistry are those well known and commonlyemployed in the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “active ingredient” refers to recombinant human insulinisophane, recombinant human regular insulin and other insulins.

As used herein, amino acids are represented by the full name thereof, bythe three-letter code as well as the one-letter code correspondingthereto, as indicated in the following table:

3-Letter 1-Letter Full Name Code Code Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic Acid Asp D Cysteine Cys C Cystine Cys-Cys C-CGlutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

The term “lower” means the group it is describing contains from 1 to 6carbon atoms.

The term “alkyl”, by itself or as part of another substituent means,unless otherwise stated, a straight, branched or cyclic chainhydrocarbon having the number of carbon atoms designated (i.e. C₁-C₆means one to six carbons) and includes straight, branched chain orcyclic groups. Examples include: methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, cyclohexyl andcyclopropylmethyl. Most preferred is (C₁-C₃) alkyl, particularly ethyl,methyl and isopropyl.

The term “alkylene”, by itself or as part of another substituent means,unless otherwise stated, a straight, branched or cyclic chainhydrocarbon having two substitution sites, e. g., methylene (—CH₂—),ethylene (—CH₂CH₂—), isopropylene (—CH(CH₃)═CH₂), etc.

The term “aryl”, employed alone or in combination with other terms,means, unless otherwise stated, a cyclic carbon ring structure, with orwithout saturation, containing one or more rings (typically one, two orthree rings) wherein such rings may be attached together in a pendantmanner, such as a biphenyl, or may be fused, such as naphthalene.Examples include phenyl; anthracyl; and naphthyl. The structure can haveone or more substitution sites where functional groups, such as alcohol,alkoxy, amides, amino, cyanides, halogen, and nitro, are bound.

The term “arylloweralkyl” means a functional group wherein an aryl groupis attached to a lower alkylene group, e.g., —CH₂CH₂-phenyl.

The term “alkoxy” employed alone or in combination with other termsmeans, unless otherwise stated, an alkyl group or an alkyl groupcontaining a substituent such as a hydroxyl group, having the designatednumber of carbon atoms connected to the rest of the molecule via anoxygen atom, such as, for example, —OCHOH—, —OCH₂OH, methoxy (—OCH₃),ethoxy (—OCH₂CH₃), 1-propoxy (—OCH₂CH₂CH₃), 2-propoxy (isopropoxy),butoxy (—OCH₂CH₂CH₂CH₃), pentoxy (—OCH₂CH₂CH₂CH₂CH₃), and the higherhomologs and isomers.

The term “acyl” means a functional group of the general formula—C(═O)—R, wherein —R is hydrogen, hydrocarbyl, amino or alkoxy. Examplesinclude acetyl (—C(═O)CH₃), propionyl (—C(═O)CH₂CH₃), benzoyl(—C(═O)C₆H₅), phenylacetyl (—C(═O)CH₂C₆H₅), carboethoxy (—CO₂ CH₂CH₃),and dimethylcarbamoyl (—C(═O)N(CH₃)₂).

The terms “halo” or “halogen” by themselves or as part of anothersubstituent mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

The term “heterocycle” or “heterocyclyl” or “heterocyclic” by itself oras part of another substituent means, unless otherwise stated, anunsubstituted or substituted, stable, mono- or multicyclic heterocyclicring system comprising carbon atoms and at least one heteroatom selectedfrom the group comprising N, O, and S, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogen atom maybe optionally quatemized. The heterocyclic system may be attached,unless otherwise stated, at any heteroatom or carbon atom which affordsa stable structure. Examples include pyrrole, imidazole, benzimidazole,phthalein, pyridenyl, pyranyl, furanyl, thiazole, thiophene, oxazole,pyrazole, 3-pyrroline, pyrrolidene, pyrimidine, purine, quinoline,isoquinoline, carbazole, etc.

The term “chromium target molecule complex” refers to a complexcomprising a number of individual units, where each unit compriseschromium (Cr) atoms capable of accepting up to six ligands contributedby multivalent molecules, such as ligands from numerous molecules ofN-(2,6-diisopropylphenylcarbamoyl methyl) iminodiacetic acid. Theindividual units are linked to each other forming a complicatedpolymeric structure linked in a three-dimensional array. The polymericcomplex is insoluble in water but soluble in organic solvents.

The term “lipid construct” refers to a lipid and/or phospholipidparticle in which individual lipid molecules cooperatively interact tocreate a bipolar lipid membrane which encloses and isolates a portion ofthe medium in which the construct resides.

The term “amphipathic lipid” means a lipid molecule having a polar andnon-polar end.

The term “extended amphipathic lipid” means an amphipathic molecule witha structure that, when part of a lipid construct, extends from the lipidconstruct into media around the construct, and can bind or interact witha receptor.

A “complexing agent” is a compound that will form a polymeric complexwith a selected metal bridging agent, e. g. a salt of chromium,zirconium, etc., that exhibits polymeric properties where the polymericcomplex is substantially insoluble in water and soluble in organicsolvents.

By “aqueous media” is meant water or water containing buffer or salt.

By “substantially soluble” is meant that the material, such as theresultant polymeric chromium target molecule complex or other metaltargeting complexes which may be crystalline or amorphous in compositionthat are formed from complexing agents, exhibit the property of beinginsoluble in water at room temperature. Such a polymeric complex or adissociated form thereof when associated with a lipid construct matrixforms a transport agent which functions to carry and deliver insulin tohepatocytes in the liver of a warm-blooded host.

By “substantially insoluble” is meant that a polymeric complex, such asa polymeric chromium target molecule complex or other metal targetingcomplexes, exhibits the property of being insoluble in water at roomtemperature. Such a polymeric complex, which may be crystalline,amorphous in composition, or a dissociated form thereof, when associatedwith a lipid construct forms a transport agent that carries and deliversinsulin to hepatocytes in the liver.

By use of the term “associated with” is meant that the referencedmaterial is incorporated into or on the surface of, or within, the lipidconstruct matrix.

The term “insulin” refers to natural or recombinant forms of insulin,and derivatives of the aforementioned insulins. Examples of insulininclude, but are not limited to insulin lispro, insulin aspart, regularinsulin, insulin glargine, insulin zinc, human insulin zinc extended,isophane insulin, human buffered regular insulin, insulin glulisine,recombinant human regular insulin, and recombinant human insulinisophane. Also included are animal insulins, such as bovine or porcineinsulin.

The term “free insulin” refers to an insulin that is not associated witha target molecule complex.

The terms “glargine” and “glargine insulin” both refer to a recombinanthuman insulin analog which differs from human insulin in that the aminoacid asparagine at position A21 is replaced by glycine and two argininesare added to the C-terminus of the B-chain. Chemically, it is21^(A)-Gly-30^(B)a-L-Arg-30^(B)b-L-Arg-human insulin and has theempirical formula C₂₆₇H₄₀₄N₇₂O₇₈S₆ and a molecular weight of 6063. Thestructural formula of glargine insulin is provided in FIG. 11.

The term “non-glargine insulin” refers at all insulins, either naturalor recombinant that are not glargine insulin. The term includesinsulin-like moieties, including fragments of insulin molecules, thathave biological activity of insulins.

The term “recombinant human insulin isophane” refers to a human insulinthat has been treated with protamine. The structural formulas forrecombinant human insulin isophane and protamine are provided in FIG.12.

The term “at least one insulin that is not recombinant human insulinisophane insulin” refers at all insulins, either natural or recombinant,that are not recombinant human insulin isophane. The term includesinsulin-like moieties, including fragments of insulin molecules thathave biological activity of insulins.

“HDV”, or “Hepatocyte Delivery Vehicle”, is a water insoluble targetmolecule complex comprising a lipid construct matrix containing multiplelinked individual units of a structure formed by the combination of ametal bridging agent and a complexing agent. “HDV” is described in WO99/59545, Targeted Liposomal Drug Delivery System.

“HDV-glargine” is a designation for a hepatocyte targeted compositioncomprising a mixture of free glargine insulin and glargine insulinassociated with a water insoluble target molecule complex, wherein thecomplex comprises multiple linked individual units of chromium andN-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid, formed bythe combination of a metal bridging agent and a complexing agent, and alipid construct matrix.

“HDV-NPH” is a designation for a hepatocyte targeted compositioncomprising a mixture of free recombinant human insulin isophane, freenon-humulin insulin, and recombinant human insulin isophane andnon-humulin insulin that are associated with a water insoluble targetmolecule complex, wherein the complex comprises multiple linkedindividual units of chromium andN-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid, formed bythe combination of a metal bridging agent and a complexing agent, and alipid construct matrix.

The term “bioavailability” refers to a measurement of the rate andextent that insulin reaches the systemic circulation and is available atthe sites of action.

The term “isoelectric point” refers to the pH at which theconcentrations of positive and negative charges on the protein are equaland, as a result, the protein will express a net zero charge. At theisoelectric point, a protein will exist almost entirely in the form of azwitterion, or hybrid between forms of the protein. Proteins are leaststable at their isoelectric points, and are more easily coagulated orprecipitated at this pH. However, proteins are not denatured uponisoelectric precipitation since this process is essentially reversible.

As the term is used herein, “to modulate” or “modulation of” abiological or chemical process or state refers to the alteration of thenormal course of the biological or chemical process, or changing thestate of the biological or chemical process to a new state that isdifferent than the present state. For example, modulation of theisoelectric point of a polypeptide may involve a change that increasesthe isolelectric point of the polypeptide. Alternatively, modulation ofthe isoelectric point of a polypeptide may involve a change thatdecreases the isolelectric point of a polypeptide.

“Statistical structure” denotes a structure formed from molecules thatcan migrate from one lipid construct to another and the structure ispresent in a plurality of particle sizes that can be represented by aGaussian distribution.

“Multi-dentate binding” is a chemical binding process that utilizesmultiple binding sites within the lipid construct, such as celluloseacetate hydrogen phthalate, phospholipids and insulin. These bindingsites promote hydrogen bonding, ion-dipole and dipole-dipoleinteractions where the individual molecules work in tandem to formnon-covalent associations that serve to bind or connect two or moremolecules.

As used herein, to “treat” means reducing the frequency with whichsymptoms of a disease, disorder, or adverse condition, and the like, areexperienced by a patient.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “physiologically acceptable” means that theingredient is not deleterious to the subject to which the composition isto be administered.

Description of the Invention—Composition

Lipid Construct

A depiction of an insulin binding lipid construct comprising insulin, anamphipathic lipid and an extended amphipathic lipid is shown in FIG. 1.The extended amphipathic lipid, also known as a receptor bindingmolecule, comprises proximal, medial and distal moieties, wherein theproximal moiety connects the extended lipid molecule to the construct,the distal moiety targets the construct to a receptor displayed by ahepatocyte, and the medial moiety connects the proximal and distalmoieties. Suitable amphipathic lipids generally comprise a polar headgroup and non-polar tail group that are attached to each other through aglycerol-backbone.

Suitable amphipathic lipids include1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine, cholesterol, cholesterololeate, dicetyl phosphate, 1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](sodium salt),triethylammonium 2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate and a mixture of any of the foregoing lipidsor appropriate derivative of these lipids.

In an embodiment, amphipathic lipid molecules include1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetylphosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(CapBiotinyl); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt),triethylammonium 2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate and a mixture of any of the foregoinglipids.

The extended amphipathic lipid molecule, also know as a receptor bindingmolecule, comprises proximal, medial and distal moieties. The proximalmoiety connects the extended lipid molecule to the construct, and thedistal moiety targets the construct to a receptor displayed by ahepatocyte. The proximal and distal moieties are connected through amedial moiety. The composition of various receptor binding molecules isdescribed below. Within a lipid construct, hepatocyte receptor bindingmolecules from one or more of the groups listed below can be present tobind the construct to receptors in the hepatocytes.

One group of hepatocyte receptor binding molecules comprises a terminalbiotin or iminobiotin moiety, as well as derivatives thereof. Thestructural formulas of biotin, iminobiotin, carboxybiotin and biocytinare shown in Table 1.

TABLE 1 1 1,2-distearoyl-sn-glycero-3- phosphocholine2,3-bis(stearoyloxy)propyl 2-(trimethylammonio)ethyl phosphate

2 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine2,3-bis(palmitoyloxy)propyl 2-(trimethylammonio)ethyl phosphate

3 1,2-dimyristoyl-sn-glycero- 3-phosphocholine 2,3-bis(tetradecanoyloxy)propyl 2-(trimethylammonio) ethyl phosphate

4 Cholesterol 10,13-dimethyl-17- (6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol

These molecules can be attached to a phospholipid molecule using avariety of techniques to create lipid anchoring molecules that can beintercalated into a lipid construct. These hepatocyte receptor bindingmolecules comprise an anchoring portion located in the proximal positionto the lipid construct. The anchor portion comprises two lipophilichydrocarbon chains that can associate and bind with other lipophilichydrocarbon chains on phospholipid molecules within the lipid construct.

In a preferred embodiment, a second group of hepatocyte receptor bindingmolecules comprises a terminal biotin or iminobiotin moiety located inthe distal position from the lipid construct. The structures of suchcompounds are given in Table 2.

TABLE 2 1 Biotin 5-((3aS,6aR)-2- oxohexahydro-1H- thieno[3,4-d]imidazol-4- yl)pentanoic acid

2 Iminobiotin 5-((3aS,6aR)-2- iminohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanoic acid

3 Carboxybiotin 5-((3aS,6aR)-1- (carboxymethyl)-2- oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl) pentanoic acid

4 Biocytin 2-amino-6-(5- ((3aS,6aR)-2- oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanamido) hexanoic acid

Both biotin and iminobiotin contain a mildly lipophilic bicyclic ringstructure attached to a five-carbon valeric acid chain at the 4-carbonposition on the bicyclic ring. In an embodiment, L-lysine amino acid maybe covalently bound to the valeric acid C-terminal carboxyl functionalgroup by reacting the carboxyl group on valeric acid with either theN-terminal α-amino group or the ε-amino group of L-lysine. This couplingreaction is performed using carbodiimide conjugation methods and resultsin the formation of an amide bond between L-lysine and biotin, asillustrated in FIG. 2.

A third group of hepatocyte receptor binding molecules compriseiminobiotin, carboxybiotin and biocytin with the valeric acid side chainattached via an amide bond to either the α-amino group or the ε-aminogroup of the amino acid L-lysine. A preferred embodiment usesiminobiotin in forming an iminobiocytin moiety as shown in FIG. 3.During synthesis of the hepatocyte receptor binding molecule, theα-amino group of iminobiocytin can react with the activated esterbenzoyl thioacetyl triglycine-sulfo-N-hydroxysuccinimide(BTA-3gly-sulfo-NHS) to form the active hepatocyte binding molecule(BTA-3gly-iminobiocytin) as shown in FIG. 4. BTA-3gly-iminobiocytinfunctions as a molecular spacer that ultimately expresses an activenucleophilic sulfhydral functional group that can be used in subsequentcoupling reactions. The spacer is located in the medial position inrelation to the lipid construct and allows the terminal iminobiocytinmoiety to extend approximately thirty angstroms from the surface of thelipid construct to develop an optimal and non-restricted orientation ofiminobiocytin for binding to the hepatocyte receptor. The medial spacercan include other derivatives that provide the correct stereo-chemicalorientation for the terminal biotin moiety. The main function of themedial spacer is to properly and covalently connect the proximal anddistal moieties in a linear array.

The BTA-3gly-sulfo-NHS portion of the hepatocyte receptor bindingmolecule can be synthesized by a number of means and in subsequent stepsbe linked to biocytin or iminobiocytin. The initial step comprisesadding benzoyl chloride to thioacetic acid to form by nucleophilicaddition a protective group for the active thio functionality. Theproducts of the reaction are the benzoyl thioacetic acid complex andhydrochloric acid, as shown in FIG. 5. Additional steps in the synthesisinvolve reacting benzoyl thioacetic acid with sulfo-N-hydroxysuccinimideusing dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a coupling agent to form benzoyl thioacetylsulfo-N-hydroxysuccinimide (BTA-sulfo-NHS), as depicted in FIG. 5.Benzoyl thioacetyl sulfo-N-hydroxysuccinimide is then reacted with theamino acid polymer (glycine-glycine-glycine). Following nucleophilicattack by the α-amino group of triglycine, benzoyl thioacetyl triglycine(BTA-3gly) is formed while the sulfo-N-hydroxysuccinimide leaving groupis solubilized by aqueous media, as shown in FIG. 5. Benzoyl thioacetyltriglycine is again reacted with dicyclohexylcarbodiimide or1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to form an ester bondwith sulfo-N-hydroxysuccinimide, as shown in FIG. 6. Thesulfo-N-hydroxysuccinimide ester of activated benzoyl thioacetyltriglycine (BTA-3gly-sulfo-NHS) is then reacted with the α-amino groupof the L-lysine functionality of biocytin or iminobiocytin to form thehepatocyte receptor binding moiety, the extended amphipathic lipidmolecule of benzoyl thioacetyl triglycine-iminobiocytin(BTA-3gly-iminobiocytin) illustrated in FIG. 7.

A second major coupling reaction for the synthesis of an hepatocytereceptor binding molecule is illustrated where benzoyl thioacetyltriglycine iminobiocytin is covalently attached through a thioether bondto a N-para-maleimidophenylbutyrate phosphatidylethanolamine, apreferred phospholipid anchoring molecule. This reaction results in amolecule that provides the correct molecular spacing between theterminal iminobiocytin ring and the lipid construct. An entire reactionscheme for forming a hepatocyte receptor binding molecule that functionsas an extended amphipathic lipid molecule is depicted in FIG. 8. Priorto reacting benzoyl thioacetyl triglycine iminobiocytin withN-para-maleimidophenylbutyrate phosphatidylethanolamine to form athioether linkage, the benzoyl protecting group is removed by heating inorder to expose the free sulfhydral functionality. The reaction shouldbe performed in an oxygen free environment to minimize oxidation of thesulfhydrals to the disulfide. Further oxidation could lead to theformation of a sulfone, sulfoxide, sulfenic acid or sulfonic acidderivative.

In an embodiment, the anchoring moiety of the molecule contains a pairof acyl hydrocarbon chains that form a lipid portion of the molecule.This portion of the molecule is non-covalently bound within the lipiddomains of the lipid construct. In an embodiment the anchoring moiety isproduced from is N-para-maleimidophenylbutyratephosphatidylethanolamine. Other anchoring molecules may be used. In anembodiment, anchoring molecules can include thio-cholesterol,cholesterol oleate, dicetyl phosphate;1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt),and mixtures, thereof. The entire molecular structure of the fullydeveloped lipid anchoring and hepatocyte receptor binding moleculedesignated LA-HRBM is shown in FIG. 8.

A fourth group of hepatocyte receptor binding molecule comprisesamphipathic organic molecules having both a water-soluble moiety and awater-insoluble moiety. The water-insoluble moiety reacts with a medialor connector moiety by coordination and bioconjugation chemicalreactions, while the water-insoluble moiety binds to the hepatocytebinding receptor in the liver. The molecule contains a distal componentcomprising either by a non-polar derivatized benzene ring structure,such as a 2,6-diisopropylbenzene derivative, or by a lipophilicheterobicyclic ring structure. The entire hepatocyte receptor bindingmolecule possesses fixed or transient charges, either positive ornegative, or various combinations thereof. These molecules contain atleast one carbonyl group located equal to or less than, but not greaterthan, approximately 13.5 angstroms from the terminal end of the distalmoiety, and at least one carbamoyl moiety containing a secondary amineand carbonyl group. The presence of a carbamoyl moiety or moietiesenhances the molecular stability of the organic molecule. A plurality ofsecondary amines can be present within the molecule. These secondaryamines contain a pair of unshared electrons allowing for ion-dipole anddipole-dipole bonding interactions with other molecules within theconstruct. These amines enhance molecular stability and provide apartially created negative charge that interacts with the distal moietyto promote hepatocyte receptor binding and specificity. An example ofthis group of receptor binding molecules ispolychromium-poly(bis)-[N-(2,6-(diisopropylphenyl)carbamoyl methyl)iminodiacetic acid]. In an embodiment, chromium III is located in the medialposition of the hepatocyte receptor binding molecule. The proximalmoiety of the hepatocyte specific binding molecule contains hydrophobicand/or non-polar structures that allow the molecules to be intercalatedinto, and subsequently bound within, the lipid construct. The medial andproximal moieties also allow for the correct stereo-chemical orientationof the distal portion of the hepatocyte receptor binding molecule.

The structure and properties of the lipid construct are governed by thestructure of the lipids and interaction between lipids. The structure ofthe lipids is governed primarily by covalent bonding. Covalent bondingis the molecular bonding force necessary to retain the structuralintegrity of the molecules comprising the individual constituents of thelipid construct. Through non-covalent interactions between lipids, thelipid construct is maintained in a three-dimensional conformation.

The non-covalent bond can be represented in general terms by anion-dipole or induced ion-dipole bond, and by the hydrogen bondsassociated with the various polar groups on the head of the lipid.Hydrophobic bonds and van der Waal's interactions can be generatedthrough induced dipole associations between the lipid acyl chains. Thesebonding mechanisms are transient in nature and result in a bond-makingand bond breaking process that occurs in a sub-femtosecond timeinterval. For example, van der Waal's interaction arises from amomentary change in dipole moment arising from a brief shift of orbitalelectrons to one side of one atom or molecule, creating a similar shiftin adjacent atoms or molecules. The proton assumes a δ⁺ charge and thesingle electron a δ⁻ charge, thus forming a dipole. Dipole interactionsoccur with great frequency between the hydrocarbon acyl chains ofamphipathic lipid molecules. Once individual dipoles are formed they canmomentarily induce new dipole formation in neighboring atoms containinga methylenic (—CH₂—) functionality. A plurality of transiently induceddipole interactions are formed between acyl lipid chains throughout thelipid construct. These induced dipole interactions last for only afraction of a femtosecond (1×10⁻¹⁵ sec) but exert a strong force whenfunctioning collectively. These interactions are constantly changing andhave a force approximately one-twentieth the strength of a covalentbond. They are nevertheless responsible for transient bonding betweenstable covalent molecules that determine the three-dimensionalstatistical structure of the construct and the stereo-specific molecularorientation of molecules within the lipid construct.

As a consequence of these induced-dipole interactions, the structure ofthe lipid construct is maintained by the exchange of lipid componentsbetween constructs. While the composition of the individual componentsof the construct is fixed, individual components of lipid constructs aresubject to exchange reactions between constructs. These exchanges areinitially governed by zero-order kinetics when a lipid component departsfrom a lipid construct. After the lipid component is released from thelipid construct, it may be recaptured by a neighboring lipid construct.The recapture of the released component is controlled by second-orderreaction kinetics, which is affected by the concentration of thereleased component in aqueous media around the construct capturing thecomponent and the concentration of the lipid construct which iscapturing the released component.

Examples of extended amphipathic lipids, along with their respectiveidentifiers, shown in Table 3 along with their chemical names, are:N-hydroxysuccinimide (NHS) biotin [1]; sulfo-NHS-biotin [2];N-hydroxysuccinimide long chain biotin [3], sulfo-N-hydroxysuccinimidelong chain biotin [4]; D-biotin [5]; biocytin [6];sulfo-N-hydroxysuccinimide-S—S-biotin [7]; biotin-BMCC [8]; biotin-HPDP[9]; iodoacetyl-LC-biotin [10]; biotin-hydrazide [11];biotin-LC-hydrazide [12]; biocytin hydrazide [13]; biotin cadaverine[14]; carboxybiotin [15]; photobiotin [16]; ρ-aminobenzoyl biocytintrifluoroacetate [17]; ρ-diazobenzoyl biocytin [18]; biotin DHPE [19];biotin-X-DHPE [20]; 12-((biotinyl)amino)dodecanoic acid [21];12-((biotinyl)amino)dodecanoic acid succinimidyl ester [22]; S-biotinylhomocysteine [23]; biocytin-X [24]; biocytin x-hydrazide [25];biotinethylenediamine [26]; biotin-XL [27]; biotin-X-ethylenediamine[28]; biotin-XX hydrazide [29]; biotin-XX-SE [30]; biotin-XX, SSE [31];biotin-X-cadaverine [32]; α-(t-BOC)biocytin [33];N-(biotinyl)-N′-(iodoacetyl)ethylenediamine [34]; DNP-X-biocytin-X-SE[35]; biotin-X-hydrazide [36]; norbiotinamine hydrochloride [37];3-(N-maleimidylpropionyl) biocytin [38]; ARP [39]; biotin-1-sulfoxide[40]; biotin methyl ester [41]; biotin-maleimide [42];biotin-poly(ethyleneglycol)amine [43]; (+) biotin 4-amidobenzoic acidsodium salt [44]; Biotin 2-N-acetylamino-2-deoxy-β-D-glucopyranoside[45]; Biotin-α-D-N-acetylneuraminide [46]; Biotin-α-L-fucoside [47];Biotin lacto-N-bioside [48]; Biotin-Lewis-A trisaccharide [49];Biotin-Lewis-Y tetrasaccharide [50]; Biotin-α-D-mannopyranoside [51];biotin 6-O-phospho-α-D-mannopyranoside [52]; andpolychromium-poly(bis)-[N-(2,6-(diisopropylphenyl) carbamoylmethyl)imino]diacetic acid [53].

TABLE 3 1 N-hydroxysuccinimide (NHS) biotin 2,5-dioxopyrrolidin-1-yl 5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidaxol-4-yl) pentanoate

2 sulfo-NHS-biotin sodium 2,5-dioxo-3- (trioxidanylthio)pyrrolidin-l-yl5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl) pentanoate

3 N-hydroxysuccinimide long chain biotin 2,5-dioxopyrrolidin-1-yl 6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoate

4 sulfo-N-hydroxysuccinimide long chain biotin sodium 2,5-dioxo-3-(trioxidanylthio)pyrrolidin-1-yl 6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)pentanamido) hexanoate

5 D-biotin 5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl)pentanoic acid

6 Biocytin 2-amino-6-(5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido) hexanoic acid

7 sulfo-N-hydroxysuccinimide-S- S-biotin sodium 2,5-dioxo-3-(trioxidanylthio)pyrrolidin-1-yl 3-((2-(4-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)butylamino)ethyl)disulfanyl)propanoate

8 biotin-BMCC 4-((2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)methyl)-N-(4-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)butyl) cyclohexanecarboxamide

9 biotin-HPDP 5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)- N-(6-(3-(pyridin-2-yldisulfanyl)propanamido)hexyl)pentanamide

10 iodoacetyl-LC-biotin N-(6-(2-iodoacetamido)hexyl)-5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4- yl)pentanamide

11 biotin-hydrazide 5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4- yl)pentanehydrazide

12 biotin-LC-hydrazide N-(6-hydrazinyl-6-oxohexyl)-5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanamide

13 biocytin hydrazide N-(5-amino-6-hydrazinyl-6-oxohexyl)-5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide

14 biotin cadaverine N-(5-aminopentyl)-5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamide

15 Carboxybiotin (3aS,6aR)-4-(4-carboxybutyl)-2-oxohexahydro-1H-thieno[3,4- d]imidazole-1-carboxylic acid

16 Photobiotin N-(3-((3-(4-azido-2- nitrophenylamino)propyl)(methyl)amino)propyl)-5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide

17 ρ-aminobenzoyl biocytin trifluoroacetate 2-(4-aminobenzamido)-6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoic acid 2,2,2-trifluoroacetate

18 ρ-diazobenzoyl biocytin 4-(1-carboxy-5-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanamido)pentylcarbamoyl) benzenediazonium chloride

19 biotin DHPE triethylammonium 2,3-diacetoxypropyl 2-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate

20 biotin-X-DHPE triethylammonium 2,3-diacetoxypropyl 2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate

21 12-((biotinyl)amino)dodecanoic acid 12-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)pentanamido) dodecanoic acid

22 12-((biotinyl)amino)dodecanoic acid succinimidyl ester2,5-dioxopyrrolidin-1-yl 12-(5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamido)dodecanoate

23 S-biotinyl homocysteine 4-mercapto-2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanamido) butanoic acid

24 biocytin-X 2-amino-6-(6-(5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido) hexanamido)hexanoic acid

25 biocytin x-hydrazide N-(5-amino-6-hydrazinyl-6-oxohexyl)-6-(5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido) hexanamide

26 Biotinethylenediamine N-(2-aminoethyl)-5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanamide

27 biotin-X 6-(5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido) hexanoic acid

28 biotin-X-ethylenediamine N-(2-aminoethyl)-6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamide

29 biotin-XX hydrazide N-(6-hydrazinyl-6-oxohexyl)-6-(5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamide

30 biotin-XX-SE 2,5-dioxopyrrolidin-l-yl 6-(6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido) hexanoate

31 biotin-XX.SSE sodium 2,5-dioxo-1-(6-(6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido) hexanoyloxy)pyrrolidine-3- sulfonate

32 biotin-X-cadaverine 5-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)pentanamido)hexanamido)pentan-1-aminium 2,2,2-trifluoroacetate

33 α-(t-BOC)biocytin 2-(tert-butoxycarbonylamino)-6-(5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoic acid

34 N-(biotinyl)-N′- (iodoacetyl)ethylenediamineN-(2-(2-iodoacetamido)ethyl)-5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamide

35 DNP-X-biocytin-X-SE 2,5-dioxopyrrolidin-1-yl 2-(6-(6-(2,4-dinitrophenylamino) hexanamido)hexanamido)-6-(6-(5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido) hexanoate

36 biotin-X-hydrazide N-(6-hydrazinyl-6-oxohexyl)-5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanamide

37 norbiotinamine hydrochloride 4-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) butan-1-aminium chloride

38 3-(N-maleimidylpropionyl) biocytin 2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-6-(5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamido)hexanoic acid

39 ARP; N′-(2-(aminooxy)acetyl)-5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanehydrazide

40 biotin-1-sulfoxide 5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanoic acid sulfoxide

41 biotin methyl ester methyl 5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)pentanoate

42 biotin-maleimide 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-(5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl) hexanehydrazide

43 Biotin-poly(ethyleneglycol) amine aminomethyl polyethylene 5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanoate

44 (+) biotin 4-amidobenzoic acid sodium salt sodium 4-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno [3,4-d]imidazol-4-yl) pentanamido)benzoate

45 Biotin 2-N-acetylamino-2- deoxy-β-D-glucopyranoside((2R,5S)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro- 2H-pyran-2-yl)methyl 5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanoate

46 Biotin-α-D-N-acetylneuraminide (2S,5R)-5-acetamido-4-hydroxy-3,3,4,5,6-pentamethyl-2-((5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanoyloxy)methyl)-6-(1,2,3-trihydroxypropyl)tetrahydro- 2H-pyran-2-carboxylic acid

47 Biotin-α-L-fucoside ((2R,5S)-3,4,5-trihydroxy- 2,3,4,5,6,6-hexamethyltetrahydro-2H-pyran- 2-yl)methyl 5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanoate

48 Biotin lacto-N-bioside See end of table for name

49 Biotin-Lewis-A trisaccharide See end of table for name

50 Biotin-Lewis-Y tetrasaccharide See end of table for name

51 Biotin-α-D-mannopyranoside ((1R,4R)-2,3,4-trihydroxy-5-(hydroxymethyl)-1,2,3,4,5- pentamethylcyclohexyl)methyl5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-dlimidazol-4-yl) pentanoate

52 biotin 6-O-phospho-α-D- mannopyranoside ((2R,5S)-3,4,5-trihydroxy-2,3,4,5,6-pentamethyl-6- (phosphonooxymethyl)tetrahydro-2H-pyran-2-yl)methyl 5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanoate

53 polychromium-poly(bis)- [N-(2,6-(diisopropylphenyl) carbamoylmethyl)imino diacetic acid]

Names of Compounds 48-50.

-   48.    ((2R,5S)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-2,3,4,6-tetramethyl-4-((((2S,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro-2H-pyran-2-yl)methoxy)methyl)    tetrahydro-2H-pyran-2-yl)methyl    5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate    ((2R,5S)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-2,3,4,6-tetramethyl-4-((((2S,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro-2H-pyran-2-yl)methoxy)methyl)    tetrahydro-2H-pyran-2-yl)methyl    5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate-   49.    (2R,3R,5S)-5-((((2S,3S,5S)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-2,4,6-trimethyl-4-((((2S,5R)-3,4,5-trihydroxy-6-    (hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro-2H-pyran-2-yl)methoxy)    methyl)tetrahydro-2H-pyran-2-yl)methoxy)methyl)-3,4-dihydroxy-2,4,5,6,6-pentamethyltetrahydro-2H-pyran-2-yl    5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate-   50.    (2S,5S)-3-acetamido-4-((((2R,5S)-5-((((2R,5S)-4,5-dihydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyl-3-((((2S,5S)-3,4,5-    trihydroxy-2,3,4,5,6,6-hexamethyltetrahydro-2H-pyran-2-yl)methoxy)methyl)tetrahydro-2H-pyran-2-yl)methoxy)    methyl)-3,4-dihydroxy-2,3,4,5,6,6-hexamethyltetrahydro-2H-pyran-2-yl)methoxy)methyl)-5-hydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro-2H-pyran-2-yl    5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate    Structure of iminobiotin compounds are not shown in Table 3. The    iminobiotin structures are analogs of the biotin structure where the    biotin group is replaced by a an iminobiotin group. An example is    shown below with the analogs N-hydroxysuccinimide biotin and    N-hydroxysuccinimide iminobiotin.

In an embodiment, a cellulose acetate hydrogen phthalate polymer isincorporated into the lipid construct where it can bind to hydrophilicfunctional groups on the insulin molecule and protect insulin fromhydrolytic degradation. Cellulose acetate hydrogen phthalate comprisestwo glucose molecules linked beta (1→4) in a polymeric arrangement inwhich some of the hydrogen atoms on the hydroxyl groups of the polymerare replaced by an acetyl functionality (a methyl group bound to acarbonyl carbon) or a phthalate group (represented by a benzene ringwith two carboxyl groups in the first and second positions of thebenzene ring). The structural formula of cellulose acetate hydrogenphthalate polymer is shown in FIG. 9. Only one carboxyl group on thephthalate ring structure is involved in a covalent ester linkage to thecellulose acetate molecule. The other carboxyl group, which contains acarbonyl carbon and a hydroxyl functionality, participates in hydrogenbonding with neighboring negative and positive charged dipoles residingon insulin and various lipid molecules.

In an embodiment, cellulose acetate hydrogen phthalate polymer interactswith the lipids through ion-dipole bonding with1,2-distearoyl-sn-glycero-3-phosphocholine phosphate and dicetylphosphate molecules. The ion-dipole bonding occurs between the δ⁺hydrogen on the hydroxyl groups of cellulose and the negatively chargedoxygen atom on the phosphate moiety of the phospholipid molecules. Thefunctional groups with the largest role in the ion-dipole interactionare the negatively charged oxygen atoms on the phosphate groups of thephospholipid molecules, hydrogen atoms on the hydroxyl groups and thehydrogen atoms on amide bonds of the insulin molecules. Negativelycharged functional groups form sites for ion-dipole interactions and forreacting with the δ⁺ hydrogen atom on individual hydroxyl groups and thehydroxyl groups of the carboxyl functionalities on cellulose acetatehydrogen phthalate. Ion-dipoles can be formed between the positivelycharged quaternary amines on the phosphocholine functionalities and theδ⁻carbonyl oxygen found on cellulose acetate hydrogen phthalate andinsulin. Sugar molecules comprising branched hydrophilic structures ininsulin can participate in hydrogen bonding and ion-dipole interactions.

The molecular configuration and the size of the polymer (with anapproximate molecular weight of 15,000 or more) enables celluloseacetate hydrogen phthalate to coat individual phospholipid molecules ofthe lipid construct in the region of the hydrophilic head group. Thiscoating protects insulin within the lipid construct from the acid milieuof the stomach. There are several ways that cellulose acetate hydrogenphthalate can be attached to the surface of molecules within the lipidconstruct. A preferred means of linking cellulose acetate hydrogenphthalate to the surface of the lipid construct is to attach thepolymeric cellulosic species to a tail of an insulin molecule thatpresents a sugar that projects from the surface of the lipid construct.This protects the insulin proteinaceous tails from enzymatic hydrolysis.

An extended amphipathic lipid comprises a variety of multi-dentatebinding sites for attachment to the receptor. Multi-dentate binding, asdefined herein, requires a plurality of potential binding sites on thesurface of insulin and its accompanying sugar moieties, as well as onthe lipid construct that can interface with carbonyl, carboxyl andhydroxyl functional groups on the cellulose acetate hydrogen phthalatepolymer. This enables the cellulose acetate hydrogen phthalate polymerto bind to a plurality of hydrophilic regions not only on the lipidconstruct but also on molecules of insulin in order to establish ashield of hydrolytic protection for the lipid construct. In this mannerboth insulin and the lipid construct are protected from the acidenvironment of the stomach following oral administration of the insulindosage form. Even though cellulose acetate hydrogen phthalate covers orshields individual lipid molecules within and on the surface of thelipid construct while passing through the stomach, once the constructmigrates to the alkaline region of the small intestine, celluloseacetate hydrogen phthalate is hydrolytically degraded. After celluloseacetate hydrogen phthalate is removed from the surface of the moleculesof the lipid construct, a lipid anchoring-hepatocyte receptor bindingmolecule, such as1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl),becomes exposed and then is available to bind with the receptor. Theemployment of a cellulose acetate hydrogen phthalate coating on insulinand the lipid construct is needed to ensure that a greaterbioavailability of insulin is achieved.

Target Molecule Complex

In an embodiment, the lipid construct comprises a target moleculecomplex comprising multiple linked individual units formed by complexinga bridging component with a complexing agent. The bridging component isa water soluble salt of a metal capable of forming a water-insolublecoordinated complex with a complexing agent. A suitable metal isselected from the transition and inner transition metals or neighbors ofthe transition metals. The transition and inner transition metals fromwhich the metal are selected from: Sc (scandium), Y (yttrium), La(lanthanum), Ac (actinium), the actinide series; Ti (titanium), Zr(zirconium), Hf (hafnium), V (vanadium), Nb (niobium), Ta (tantalum), Cr(chromium), Mo (molybdenum), W (tungsten), Mn (manganese), Tc(technetium), Re (rhenium), Fe (iron), Co (cobalt), Ni (nickel), Ru(ruthenium), Rh (rhodium), Pd (palladium), Os (osmium), Ir (iridium),and Pt (platinum). The neighbors of the transition metals from which themetal can be selected are: Cu (copper), Ag (silver), Au (gold), Zn(zinc), Cd (cadmium), Hg (mercury), Al (aluminum), Ga (gallium), In(indium), Tl (thallium), Ge (germanium), Sn (tin), Pb (lead), Sb(antimony) and Bi (bismuth), and Po (polonium). Examples of metalcompounds useful as bridging agents include chromium chloride (III)hexahydrate; chromium (III) fluoride tetrahydrate; chromium (III)bromide hexahydrate; zirconium (IV) citrate ammonium complex; zirconium(IV) chloride; zirconium (IV) fluoride hydrate; zirconium (IV) iodide;molybdenum (III) bromide; molybdenum (III) chloride; molybdenum (IV)sulfide; iron (III) hydrate; iron (III) phosphate tetrahydrate, iron(III) sulfate pentahydrate, and the like.

The complexing agent is a compound capable of forming a water insolublecoordinated complex with a bridging component. There are severalfamilies of suitable complexing agents.

A complexing agent can be selected from the family of iminodiaceticacids of the formula (1) where R₁ is loweralkyl, aryl, arylloweralkyl,and a heterocyclic substituent.

Suitable compounds of the formula (1) include:

-   N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,6-diethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-isopropylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,3-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-tertiary butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3-butoxyphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;-   aminopyrrol iminodiacetic acid;-   N-(3-bromo-2,4,6-trimethylphenylcarbamoylmethyl) iminodiacetic acid;-   benzimidazole methyl iminodiacetic acid;-   N-(3-cyano-4,5-dimethyl-2-pyrrylcarbamoylmethyl) iminodiacetic acid;-   N-(3-cyano-4-methyl-5-benzyl-2-pyrrylcarbamoylmethyl) iminodiacetic    acid; and-   N-(3-cyano-4-methyl-2-pyrrylcarbamoylmethyl) iminodiacetic acid and    other derivatives of N-(3-cyano-4-methyl-2-pyrrylcarbamoylmethyl)    iminodiacetic acid of formula (2),

-   -   where R₂ and R₃ are the following:

R₂ R₃ H iso-C₄H₉ H CH₂CH₂SCH₃ H CH₂C₆H₄-p-OH CH₃ CH₃ CH₃ iso-C₄H₉ CH₃CH₂CH₂SCH₃ CH₃ C₆H₅ CH₃ CH₂C₆H₅ CH₃ CH₂C₆H₄-p-OCH₃

A complexing agent is selected from the family of imino diacidderivatives of the general formula (3), where R₄, R₅, and R₆ areindependent of each other and can be hydrogen, loweralkyl, aryl,arylloweralkyl, alkoxyloweralkyl, and heterocyclic.

Suitable compounds of the formula (3) include: N′-(2-acetylnaphthyl)iminodiacetic acid (NAIDA); N′-(2-naphthylmethyl) iminodiacetic acid(NMIDA); iminodicarboxymethyl-2-naphthylketone phthalein complexone; 3(3: 7a: 12a: trihydroxy-24-norchol anyl-23-iminodiacetic acid;benzimidazole methyl iminodiacetic acid; and N-(5,pregnene-3-p-ol-2-oylcarbamoylmethyl) iminodiacetic acid.

A complexing agent is selected from the family of amino acids of formula(4),

where R₇ is an amino acid side chain, R₈ is loweralkyl, aryl,arylloweralkyl, and R₉ is pyridoxylidene.

Suitable amino acids of the formula (4) are aliphatic amino acids,including, but not limited to: glycine, alanine, valine, leucine,isoleucine; hydroxyamino acids, including serine, and threonine;dicarboxylic amino acids and their amides, including aspartic acid,asparagine, glutamic acid, glutamine; amino acids having basicfunctions, including lysine, hydroxylysine, histidine, arginine;aromatic amino acids, including phenylalanine, tyrosine, tryptophan,thyroxine; and sulfur-containing amino acids, including cystine,methionine.

A complexing agent is selected from amino acid derivatives including,but not necessarily limited to (3-alanine-y-amino) butyric acid,O-diazoacetylserine (azaserine), homoserine, ornithine, citrulline,penicillamine and members of the pyridoxylidene class of compoundsincluding, but are not limited to: pyridoxylidene glutamate;pyridoxylidene isoleucine; pyridoxylidene phenylalanine; pyridoxylidenetryptophan; pyridoxylidene-5-methyl tryptophan;pyridoxylidene-5-hydroxytryptamine; andpyridoxylidene-5-butyltryptamine.

A complexing agent is selected from the family of diamines of thegeneral formula (6),

where R₁₀ is hydrogen, loweralkyl, or aryl; R₁₁ is loweralkylene orarylloweralky; R₁₂ and R₁₃ independently are hydrogen, loweralkyl,alkyl, aryl, arylloweralkyl, acylheterocyclic, toluene, sulfonyl ortosylate.

Some suitable diamines of the formula (6) include, but are not limitedto, ethylenediamine-N, N diacetic acid; ethylenediamine-N,N-bis(-2-hydroxy-5-bromophenyl) acetate; N′-acetylethylenediamine-N,Ndiacetic acid; N′-benzoyl ethylenediamine-N,N diacetic acid;N′-(p-toluenesulfonyl) ethylenediamine-N, N diacetic acid;N′-(p-t-butylbenzoyl) ethylenediamine-N, N diacetic acid;N′-(benzenesulfonyl) ethylenediamine-N, N diacetic acid;N′-(p-chlorobenzenesulfonyl) ethylenediamine-N, N diacetic acid;N′-(p-ethylbenzenesulfonyl ethylenediamine-N,N diacetic acid; N′-acyland N′-sulfonyl ethylenediamine-N, N diacetic acid;N′-(p-n-propylbenzenesulfonyl) ethylenediamine-N, N diacetic acid;N′-(naphthalene-2-sulfonyl) ethylenediamine-N, N diacetic acid; andN′-(2, 5-dimethylbenzenesulfonyl) ethylenediamine-N, N diacetic acid.

Other suitable complexing compounds or agents include, but are notlimited to: penicillamine; p-mercaptoisobutyric acid; dihydrothiocticacid; 6-mercaptopurine; kethoxal-bis(thiosemicarbazone); HepatobiliaryAmine Complexes, 1-hydrazinophthalazine (hydralazine); sulfonyl urea;Hepatobiliary Amino Acid Schiff Base Complexes; pyridoxylideneglutamate; pyridoxylidene isoleucine; pyridoxylidene phenylalanine;pyridoxylidene tryptophan; pyridoxylidene 5-methyl tryptophan;pyridoxylidene-5-hydroxytryptamine; pyridoxylidene-5-butyltryptamine;tetracycline; 7-carboxy-p-hydroxyquinoline; phenolphthalein; eosin Ibluish; eosin I yellowish; verograffin; 3-hydroxyl-4-formyl-pyrideneglutamic acid; Azo substituted iminodiacetic acid; hepatobiliary dyecomplexes, such as rose bengal; congo red; bromosulfophthalein;bromophenol blue; toluidine blue; and indocyanine green; hepatobiliarycontrast agents, such as iodipamide; and ioglycamic acid; bile salts,such as bilirubin; cholgycyliodohistamine; and thyroxine; hepatobiliarythio complexes, such as penicillamine; p-mercaptoisobutyric acid;dihydrothiocytic acid; 6-mercaptopurine; and kethoxal-bis(thiosemicarbazone); hepatobiliary amine complexes, such as1-hydrazinophthalazine (hydralazine); and sulfonyl urea; hepatobiliaryamino acid Schiff Base complexes, includingpyridoxylidene-5-hydroxytryptamine; andpyridoxylidene-5-butyltryptamine; hepatobiliary protein complexes, suchas protamine; ferritin; and asialo-orosomucoid; and asialo complexes,such as lactosaminated albumin; immunoglobulins, G, IgG; and hemoglobin.

The three-dimensional target molecule complex made from combiningbridging agents and complexing agents is described in WO 99/59545, whichis incorporated herein by reference. In an embodiment, the bridgingagent is a metal salt, such as chromium chloride hexahydrate, capable offorming a coordinated complex with complexing agents, such asN-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid. Thebridging agent and the complexing agents are combined to form a complexcomposed of multiple linked units in a three-dimensional array. In apreferred embodiment, the complex is composed of multiple units ofchromium (bis) [N-(2,6-(diisopropylphenyl)carbamoyl methyl)iminodiacetic acid] linked together. In an embodiment, the chromium targetmolecule complex substance is soluble in a mixture of lipids containing1,2-distearoyl-sn-glycero-3-phosphocholine, dicetyl phosphate andcholesterol. The complex is incorporated within a lipid construct formedfrom the groups of lipids previously described.

Modification of the Isoelectric Point of Insulin

The isoelectric point of a protein can affect the release anddistribution of the protein in the body of a patient treated with theprotein. By changing the isolectric point of a protein, the rate ofrelease of the protein from the site of administration may be alteredand the pharmacokinetics of the protein can be changed.

One method of altering the isolectric point of insulin is to alter itsmolecular structure by substituting or adding various amino acids. Twoexamples of altering the structure of insulin to obtain differentproperties are glargine insulin and insulin aspart. Both of theseinsulins differ in amino acid composition from recombinant human regularinsulin. Recombinant human regular insulin has an isoelectric point at5.30-5.35. Glargine insulin substitutes glycine for asparagine atposition A21 and two arginines are added at the C-terminus of the Bchain. The isoelectric points of glycine and asparagine are 5.97 and5.41, respectively. The substitution of glycine for asparagine haslittle or no effect on the isoelectric point of glargine insulin.However, the addition of two highly basic arginine amino acid residues,with isoelectric points of 10.76, significantly raises the isoelectricpoint of glargine insulin to pH 5.8-6.2.

Insulin aspart substitutes aspartic acid for praline at position B-28.The isoelectric points of aspartic acid and praline are 2.97 and 6.10,respectively. With this single acidic amino acid substitution, theisoelectric point of insulin aspart is shifted significantly toward alower, more acidic, pH.

These two examples of commercially available insulins illustrate how arelatively small number of amino acid substitutions can significantlyeither raise or lower the isoelectric points of insulin glargine orinsulin aspart with respect to recombinant human regular insulin. Byaltering the chemical properties of insulin, the bioavailability andpharmacodynamic profiles are also changed. When an insulin with amodified structure is administered to a diabetic patient in order toimprove bioavailability, the new pharmacological responses provide newtherapeutic benefits.

The isoelectric point of insulins can be modified not only by internalmolecular restructuring of the primary amino acid sequences of insulin,but also by binding charged organic molecules to insulin. The chargedorganic molecules can be bound to the surface, or within the insulinstructure. The isoelectric point of native insulin can be changed frompH 5.3 to pH 7.2 by adding between 1.0 and 1.5 mg of a mixture of highlybasic proteins to 1.0 ml of an insulin solution containing 100 units or3.65 mg of insulin/ml. Protamines are an example of a group of simple,highly basic proteins that can be can used to alter the isoelectricpoint of insulin. Protamines yield numerous basic amino acids onhydrolysis, possess a high nitrogen content and occur naturally,combined with nucleic acid, in the sperm of fish. For example, theprotamines salmine, clupeine, iridine, sturine and scombrine areisolated from salmon, herring, trout, sturgeon and mackerel sperm,respectively. These basic proteins, either individually or as a mixture,associate with insulin and increase the isoelectric point of insulin.

Compounds that alter the surface charge of insulin include derivativesof polylysine and other highly basic amino acid polymers, such aspolyornithine, polyhydroxylysine, polyarginine and polyhistidine orcombinations thereof. Other polymers include poly (arg-pro-thr)_(n) in amole ratio of 1:1:1 with a molecular weight range of a few hundred toseveral thousand or poly (DL-Ala-poly-L-lys)_(n) in a mole ratio of 6:1with a molecular weight range of a few hundred to several thousand.Histones, basic proteins that exist in several subtypes that containdifferent and varying amounts of arginine, lysine and other basic aminoacids that can bind ionically to carboxyl groups of insulin, andfragments of histones, are also used to provide a positive charge. Alsoincluded are polymers such as polyglucosamine, polygalactosamine andvarious other sugar polymers that contain a positive charge contributedby a primary amino group. Polynucleotides such as polyadenine,polycytosine or polyguanine that provide a positive charge through theionization of their primary amino group are also used. All the abovepolymeric species when bound to insulin provide an increase in positivecharge that is accompanied by an increase in the isoelectric point ofinsulin. Small amounts of these polymeric compounds, such as a fewmicrograms of polymer/ml of insulin, areadded to change the isoelectricpoint of insulin a minimal amount, generally less that one pH unit.Larger amounts, generally greater than a milligram or two, of basicorganic compounds can be added per ml of insulin at 100 units/ml toprogressively increase the isoelectric point of insulin to more than twopH units beyond its native isoelectric point.

Conversely, the isoelectric point of insulin can be lowered in a similarfashion by adding carboxylated polymers and polymeric amino acids suchas polyaspartic acid, polyglutamic acid, proteins or fragments ofproteins that contain large amounts of amino acid residues with carboxyl(COO⁻) or sulfhydral (S⁻) functional groups. Highly basic proteins canbe changed to highly acidic proteins by reacting them with anappropriate anhydride, such as acetic anhydride, to form a negativelycharged terminal acidic carboxyl group in place of a positively chargedbasic primary amino group. Other acidic polymers, such as sulfate-ladenpolymers, may be added to insulin to lower the isoelectric point ofinsulin. Sugar polymers such as polygalacturonic acid, polygluconicacid, polyglucuronic acid or polyglucaric acid that contain negativelycharged carboxyl groups can be used to lower the isoelectric point ofthe protein.

Changing the isoelectric point of an insulin alters not only the ioniccharacter of the native insulin molecule, but also the nature of theionic envelope, known as the Hemholtz double layer, that surroundsinsulin and extends into the bulk phase aqueous media around theinsulin. The ionic environment surrounding insulin tends to exist inlayers with a layer of counter-ions associated with the participatingcharged organic molecules that are bound to insulin. An electricpotential exists on modified insulin molecules that are maintained in acolloidal suspension in bulk phase media because of the presence of ionson the surface of insulin. That part of the electric potential existingbetween the layer of fixed counter ions associated with the boundorganic molecules and that of the bulk phase media is know as theelectrokinetic or zeta (ξ) potential. The zeta potential contributessignificantly to the electrical properties and stability of colloidalsystems such as insulin in aqueous media.

As a result of forming a different chemical structure by the addition ofmaterial to change the isoelectric point, the stability of the proteininsulin in colloidal suspension is inherently altered. Insulinexperiences a shift in stability at the newly modified isoelectric pointdue to a lower zeta potential. Insulin is least stable when it is in thezwitterionic, or hybrid form, where the negatively charged functionalgroups precisely balance the positively charged functional groups andcreate an overall net zero charge on the protein. Even though theoverall net charge is zero, there remain pockets of negative charge andpockets of positive charge throughout the protein structure. As the pHof a solution of insulin reaches its isoelectric point, its solubilitydecreases and insulin may precipitate from solution. During theisoelectric precipitation of insulin, the insulating and dielectricproperties of the bulk phase aqueous buffer media are overcome and theion atmosphere of the Hemholtz double layer is fractured so thatdissimilar charges between colloidal particles can associate which leadsto a protein colloidal suspension with increasing instability. Theseeffects eventually result in the coagulation and subsequentprecipitation of the protein at the isoelectric point. The ideal rangefor isoelectric precipitation is two or three pH units above or belowthe isoelectric point of insulin at pH 5.3. However, isoelectric pointsextending beyond this pH range may be formulated through the use ofinformation by one skilled in the art.

As the pH changes from the isoelectric point, solubility increase andinsulin that precipitated at the isoelectric point can be resolubilized.This occurs because as the pH is increased or decreased from theisoelectric point, there is a respective accumulation of negative charge(above the isoelectric point) or an accumulation of positive charge(below the isoelectric point) that is regulated by the pKa of therepresentative functional groups. Resolubilization occurs as the proteindevelops a greater disparity of charge thereby increasing the zetapotential of the protein which in turn improves protein stability. Theseeffects result in the redevelopment of an ionic envelope which surroundsthe protein which facilitates greater colloidal dispersion of theinsulin molecules.

The isoelectric point of native insulin, which occurs at pH 5.3, can beprogressively raised by adding proteins, peptide fragments, polymers orpolymer fragments that bind to insulin and alter the ionic character ofinsulin. The overall affect of adding basic functional groups is toraise the isoelectric point of insulin and create an insulin that has aslower onset of pharmacological action by having the insulin transitionbetween a soluble from, to an insoluble form, and then to a new solubleform. By modifying the isoelectric point of native insulin, especiallyin the presence of HDV insulin, the bioavailability of both insulinforms can be regulated.

Insulins in which the isoelectric point was altered by changing theamino acid sequence can be incorporated into a lipid construct. In anembodiment, glargine insulin is incorporated into a target moleculecomplex comprising a lipid and multiple linked individual units formedby complexing a bridging component with a complexing agent. Adescription of the target molecule complex and its components waspreviously described herein. The structure of glargine insulin isprovided in FIG. 11. Glargine insulin differs from human insulin in thatglargine insulin has a molecular structure that replaces asparagine withglycine at the C-terminal end of the A chain of human insulin and addsthe dipeptide of arginine at the C-terminal end of the B chain of humaninsulin. The isoelectric point of a compound is the pH at which theoverall charge of the compound is neutral. However, regions of negativeand positive charges still remain within the compound. The isoelectricpoint of human insulin is at pH 5.3. The isoelectric point of glargineinsulin is higher than human insulin because the amino acidsubstitutions in glargine insulin raise the isoelectric point ofglargine insulin to pH 5.8-6.2. Compounds are generally less soluble inaqueous solutions at pH ranges around the isoelectric point. A compoundis generally more soluble in aqueous systems where the pH of thesolution is approximately 1-2 pH units higher or lower than theisoelectric point. The higher isoelectric point allows glargine insulinto remain soluble in a mildly acidic environment over a broader pHrange.

A commercial form of glargine insulin, LANTUS® (insulin glargine [rDNAorigin] injection), is a sterile solution of glargine insulin for use asan injectable insulin for diabetic patients for subsequent management ofglucose levels in vivo. Glargine insulin is a recombinant human insulinanalog that is a long-acting (up to 24-hour duration of action),parenteral blood-glucose-lowering agent. LANTUS® is produced byrecombinant DNA technology utilizing a non-pathogenic laboratory strainof Escherichia coli (K12) as the production organism. LANTUS consists ofglargine insulin dissolved in a clear aqueous fluid. Each milliliter ofLANTUS (insulin glargine injection) contains 100 IU (3.6378 mg) glargineinsulin, 30 mcg zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, and water forinjection. The pH of commercially available LANTUS insulin can beadjusted by addition of aqueous solutions of acids, bases or buffersthat are physiologically compatible. LANTUS has a pH of approximately 4.

A depiction of a pharmaceutical composition that combines free insulinand insulin associated with a target molecule complex is shown in FIG.13. In an embodiment, a pharmaceutical composition may comprise two ormore insulins. The target molecule complex comprises multiple linkedindividual units formed by complexing a bridging component with acomplexing agent. The bridging component is a water soluble salt of ametal capable of forming a water-insoluble coordinated complex with acomplexing agent. A suitable metal is selected from the transition andinner transition metals or neighbors of the transition metals. Adescription of the target molecule complex and its components waspreviously described herein. In an embodiment, a pharmaceuticalcomposition comprises a mixture of free insulin and insulin associatedwith a water insoluble target molecule complex. Free insulin is notassociated with the target molecule complex and is soluble in water. Theother form of insulin in the composition is associated with a waterinsoluble target molecule complex.

Adjustment of the pH of an aqueous solution surrounding the lipidconstruct containing the target molecule complex, by the addition ofacids, bases, or buffers, results in a negative charge in the lipidconstruct structure. The pH range at which this occurs depends upon thecomposition of the lipids. A preferred lipid system is a mixture of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol anddicetylphosphate. This mixture forms a negatively charged lipidconstruct structure under physiological conditions. The lipid constructexhibits hepatocyte targeting specificity, i.e. is specific for cellularhepatocytes, thereby allowing the construct to be targeted to the liver.

It has been discovered in the present invention that when theappropriate lipid components are formulated into a water insolubletarget molecule complex using Sterile Water for Injection, USP (SWI)that has been terminally pH adjusted to pH 3.95±0.2, the overallelectronic charge on the target molecule complex is predominatelynegative. Glargine insulin has a net positive charge at pH 5.2±0.5,which is below the isoelectric point of the protein. The positive chargeon glargine insulin at pH 5.2±0.5 allows for interaction of thepositively charged portion of glargine insulin with the negativelycharge portion of the target molecule complex. This results inpositively charged glargine insulin being attracted to the negativelycharged target molecule complex. Portions of the charged glargineinsulin become associated with charges on the lipids and the chargedglargine insulin moves within the lipids, while other charged glargineinsulin molecules are sequestered within the core volume of the lipidconstruct after partitioning through the various lipid moieties of thelipid construct.

There is an equilibrium between free glargine insulin in solution andglargine insulin associated with the water insoluble target moleculecomplex. Because the interactions between glargine insulin and thetarget molecule complex involve equilibria, over time free glargineinsulin is able to further bind and partition into the lipid domainsand/or the central core volume of the water insoluble target moleculecomplex. In an embodiment, free glargine insulin can be transformed intotransitory lipid derivatives by adsorbing onto, or reacting with,individual molecules of lipid that are in equilibrium with the waterinsoluble target molecule complex. These derivatives associate with thelipids of the water insoluble target molecule complex and enter thecore-volume of the complex, thus affecting the pharmacological activityof the product.

Insulins in which the isoelectric point was altered by binding chargedorganic molecules to insulin can be incorporated into a lipid construct.In an embodiment, recombinant human insulin isophane is incorporatedinto a target molecule complex comprising a lipid and multiple linkedindividual units formed by complexing a bridging component with acomplexing agent.

The structure of recombinant human insulin isophane and protamine areprovided in FIG. 12. Recombinant human insulin isophane differs fromhuman insulin in that recombinant human insulin has been treated withprotamine such that protamine forms a coating over the insulin. Theisoelectric point of recombinant human insulin isophane (pH 7.2) ishigher than human insulin (pH 5.3) because the addition of protamine torecombinant human insulin isophane raises the isoelectric point of theprotein. The higher isoelectric point allows recombinant human insulinisophane to remain insoluble at physiological pH. The Humulin NPHinsulin product currently marketed exists as a milky suspension whererecombinant human insulin isophane settles to the bottom of the vial.

In an embodiment, a pharmaceutical composition comprises a mixture offree recombinant human insulin isophane and free recombinant humanregular insulin and recombinant human insulin isophane and recombinanthuman regular insulin that is associated with a water insoluble targetmolecule complex. Free recombinant human insulin isophane is thematerial depicted in FIG. 12. Free recombinant human insulin isophane isnot associated with the target molecule complex and is insoluble atphysiological pH of approximately 7.2, the isoelectric point of NPHinsulin. Recombinant human regular insulin is soluble at pH 7.2.

For each of the insulins, there is an equilibrium between the free formof insulin in solution or suspension and the forms of the insulinassociated with the water insoluble target molecule complex. Because theinteractions between each form of insulin and the target moleculecomplex involve equilibria, over time the free forms of the insulinsbind and partition into the lipid domains and/or the central core volumeof the water insoluble target molecule complex. In an embodiment, freerecombinant human insulin isophane and recombinant human regular insulincan be transformed into transitory lipid derivatives by adsorbing onto,or reacting with, individual molecules of lipid that are in equilibriumwith the water insoluble target molecule complex. These derivativesassociate with the lipids of the water insoluble target molecule complexand enter the core-volume of the complex, thus affecting thepharmacological activity of the product.

Description of the Invention—Method of Manufacturing the Lipid Construct

FIG. 14 demonstrates an outline for the process for manufacturing alipid construct comprising an amphipathic lipid, an extended amphipathiclipid and insulin. The manufacture of the composition comprises threeoverall steps: preparing a mixture of an amphipathic lipid and anextended amphipathic lipid, preparing a lipid construct from the mixtureof an amphipathic lipid and an extended amphipathic lipid, and combininginsulin into the lipid construct.

Lipids are produced and loaded by the methods disclosed herein, andthose methods described in U.S. Pat. Nos. 4,946,787; 4,603,044; and5,104,661, and the references cited therein. Typically, the aqueouslipid construct formulations of the invention comprise 0.1% to 10%active agent by weight (i.e. 1-10 mg drug per ml), and 0.1% to 4% lipidby weight in an aqueous solution, optionally containing salts andbuffers, in a quantity to make 100% by volume. Preferred areformulations which comprise 0.1% to 5% active agent. Most preferred is aformulation comprising 0.01% to 5% active agent by weight and up to 2%by weight of a lipid component in an amount of aqueous solutionsufficient (q. s.) to make 100% by volume.

In an embodiment, the lipid construct is prepared by the followingprocedure. Individual lipid constituents are mixed together in anorganic solvent system where the solvent had been dried over molecularsieves for approximately two hours to remove any residual water that mayhave accompanied the solvent. In an embodiment, the solvent systemcomprises a mixture chloroform and methanol in the ratio 2:1 by volume.Other organic solvents that can be easily removed from a mixture ofdried lipids also can be used. Use of a single-step addition of thelipid constituents in the initial mixing procedure obviates the need forintroducing any additional coupling reactions which would unnecessarilycomplicate the structure of the lipid construct and require additionalseparation procedures. The lipid components and the hepatocyte receptorbinding molecule are dissolved in the solvent, then the solvent isremoved under high vacuum until a dried mixture of the lipids forms. Inan embodiment, the solvent is removed under vacuum using arotoevaporator, or other methods known in the art, with slow turning atapproximately 60° C. for approximately two hours. This mixture of lipidscan be stored for further use, or used directly.

The lipid construct is prepared from the dried mixture of amphipathiclipids and an extended amphipathic lipid. The dried mixture of lipidsare added to an appropriate amount of aqueous buffered media, then themixture is swirled to form a homogeneous suspension. The lipid mixtureis then heated with mixing at approximately 80° C. for approximately 30minutes under a dry nitrogen atmosphere. The heated homogeneoussuspension is immediately transferred to a micro-fluidizer preheated toapproximately 70° C. The suspension is passed through themicrofluidizer. The suspension may require additional passes through themicrofluidizer to obtain a homogeneous lipid micro-suspension. In anembodiment a Model #M-110 EHI micro-fluidizer was used where thepressure on the first pass was approximately 9,000 psig. A second passof the lipid suspension through the micro-fluidizer may be needed toproduce a product that exhibits the properties of a homogeneous lipidmicro-suspension. This product is defined structurally andmorphologically as a three-dimensional lipid construct which contains ahepatocyte receptor binding molecule.

Insulin is loaded into the lipid constructs using one of two methods:equilibrium loading and non-equilibrium loading. Equilibrium loading ofinsulin begins when insulin is added to a suspension of the lipidconstructs. Over time, insulin molecules move into and out of the lipidconstruct. The movement is governed by partitioning equilibrium, wheremovement into the lipid construct after the initial introduction ofinsulin to the suspension.

Non-equilibrium loading of insulin into the lipid constructs localizesinsulin within the lipid construct. Following equilibrium loading offree insulin into the lipid construct, the bulk phase media thatcontains free insulin is removed. The non-equilibrium loading procedureis a vector-driven process that begins the instant the external bulkphase media is removed. The gradient potential for insulin to migrateout of the lipid constructs is eliminated when the aqueous phasecontaining insulin has been removed. The overall process results in agreater concentration of insulin within the final lipid constructbecause movement of insulin from within the construct is eliminated. Theequilibrium loading of insulin is a time-dependent phenomenon whereasthe non-equilibrium loading procedure is practically instantaneous.Non-equilibrium loading can be initiated by a variety of processes wherethe material in solution is separated from the lipid construct. Examplesof such processes include, but are not limited to: filtration, centriconfiltration, centrifugation, batch style affinity chromatography,streptavidin agarose affinity-gel chromatography or batch styleion-exchange chromatography. Any means that eliminates the gradientpotential for insulin diffusion and leakage and causes the insulin to beretained by the lipid construct can be utilized.

When using batch-style chromatography, the affinity or ion-exchange gelis mixed rapidly with the mixture of insulin and the construct. Bindingto the chromatography medium occurs rapidly and the chromatographymedium is removed from the aqueous media by decanting of the aqueousphase or by using classic filtering techniques such as the use of filterpaper and a Büchner funnel.

The lipid construct contains a discrete amount of loaded insulin locatednot only inside, but also within and on the surface of the lipidconstruct. The lipid construct created is a new and novel composition ofmatter and becomes a composition for delivering an effective amount ofinsulin as a result of non-equilibrium loading. The loading of insulininto this lipid construct and the subsequent removal of bulk phaseinsulin results in a high concentration of insulin in a lipid constructby shortening the length of time needed for removal of the externalphase media. It would be difficult to achieve this level of loadinginsulin into the construct using time-dependent procedures, such asion-exchange or gel-filtration chromatography, since these proceduresrequire a constant infusion of buffer comprising high concentrations ofinsulin. For example, loading insulin into the construct using smallscale column chromatography requires approximately twenty minutes toremove the external bulk phase media containing insulin from theconstruct containing insulin. Equilibrium conditions are reestablishedduring this time period by movement of insulin from the construct.Maintaining a high concentration of insulin in and on the lipidconstruct is one of the positive benefits of using non-equilibriumloading.

In an extension of the non-equilibrium loading process, celluloseacetate hydrogen phthalate is added to the lipid construct during thestep of loading insulin to the lipid construct after the insulin hasundergone equilibrium loading but before the non-equilibrium loadingprocess is initiated. The nature and structure of the insulin moleculeallows it to be intercalated into the lipid construct were insulin isdispersed throughout the lipid construct. Hydrophilic portions ofinsulin, as well as branched complex sugars and additional functionalgroups, extend into the bulk phase media from the surface of the lipidconstruct. These extended hydrophilic portions of insulin canparticipate in hydrogen bonding, dipole-dipole and ion-dipoleinteractions at the surface of the lipid construct with the hydroxylgroups, carboxyl groups and carbonyl functionalities of celluloseacetate hydrogen phthalate as illustrated in FIG. 9. Cellulose acetatehydrogen phthalate offers a unique means of combining with the moleculesof the lipid construct to provide an excellent shield for masking thecontents of the lipid construct from the digestive milieu of thestomach. The digestive processes in the stomach result from thehydrolytic cleavage of proteinaceous substrates by the enzyme pepsin aswell as cleavage by acid hydrolysis. The acidic environment of thestomach degrades free insulin and can hydrolyze the ester bonds thathold the acyl hydrocarbon chains to the glycerol backbone in thephospholipid molecules. Hydrolytic cleavage can also occur on eitherside of the phosphate functionality in the phosphocholine group. Thedigestive system changes from the acid region of the stomach to analkaline region of the small intestine were enzymatic action of trypsinand chymotrypsin occurs. Amino acid lysing enzymes, such as alpha aminopeptidases, can degrade proteins such as insulin from the N-terminalend. The presence of cellulose acetate hydrogen phthalate in the lipidconstruct protects insulin from hydrolytic degradation. As the alkalineenvironment of the small intestine hydrolytically degrades the celluloseacetate hydrogen phthalate shield of the lipid construct the hepatocytereceptor binding molecule becomes available to direct binding of theconstruct to the hepatocyte binding receptor. While not wishing to bebound by any particular theory, there is a synergy of hydrolyticprotection upon the addition of cellulose acetate hydrogen phthalate atthe end point of non-equilibrium loading. The protection is distributednot only to insulin and individual lipid molecules, but also to theentire lipid construct. This synergy provides collective as well asindividual molecular protection from enzymatic and acid hydrolysis.

In an embodiment, cellulose acetate hydrogen phthalate is covalentlybound to either insulin or the lipid construct using a variety ofmethods. For example, one method involves coupling the hydroxyl groupson cellulose acetate hydrogen phthalate with the amine functionalitieson either 1,2-diacyl-sn-glycero-3-phosphoethanolamine or the ε-aminogroup of the ten L-lysines in the insulin molecule utilizing the Mannichreaction.

In an embodiment, cellulose acetate hydrogen phthalate is loaded intothe lipid construct during equilibrium loading of insulin into theconstruct. The hydroxyl and carbonyl functionalities of the celluloseacetate hydrogen phthalate hydrogen bond with lipid molecules in a lipidconstruct. Hydrogen bonds between cellulose acetate hydrogen phthalateand the construct are formed concurrently as insulin is loaded underequilibrium conditions into the lipid construct creating a shield aroundinsulin and around the construct.

HDV-Insulin is recovered and recycled from aqueous media by binding itto streptavidin-agarose iminobiotin. Streptavidin covalently bound tocyanogen bromide activated agarose provides a means to separate animinobiotin-based lipid construct from insulin in the aqueous media atthe end of non-equilibrium loading of insulin into the construct. In anembodiment, an iminobiotin derivative forms the hepatocyte receptorbinding portion of the phospholipid moiety within the lipid construct.The water-soluble portion of the lipid anchoring molecule extendsapproximately 30 angstroms from the lipid surface to facilitate bindingof the hepatocyte receptor binding portion of the phospholipid moietywith a hepatocyte receptor and to aid in the attachment of the lipidconstruct to streptavidin.

Streptavidin reversibly binds to iminobiotin at pH values of 9.5 andgreater, where the uncharged guandino functional group of iminobiotinstrongly binds to one of the four binding sites on streptavidin locatedapproximately nine angstroms below the surface of the protein. A lipidconstruct containing iminobiotin is removed from buffered media byraising the pH of an aqueous mixture of the construct to pH 9.5 by theaddition of a 20 mM sodium carbonate-sodium bicarbonate buffer. At thispH, the bulk phase media contains free insulin which is reclaimed andseparated from the lipid construct using a variety of proceduresincluding to, but not limited to filtration, centrifugation orchromatography.

The mixture at pH 9.5 is then mixed with streptavidin-agarosecross-linked beads, where the construct is adsorbed onto thestreptavidin. The beads, which are approximately 120 microns indiameter, are separated from the solution by filtration. The lipidconstruct is released from the streptavidin-agarose affinity-gel byreducing the pH from pH 9.5 to pH 4.5 by the addition of a 20 mM sodiumacetate-acetic acid buffer at pH 4.5. At pH 4.5 the guandino group ofiminobiotin becomes protonated and positively charged, as shown in FIG.10. The lipid construct is released and separated from thestreptavidin-agarose bead by filtration. The streptavidin-agarose beadare reclaimed for additional usage. Thus both free insulin andstreptavidin-agarose are conserved and can be re-used.

In an embodiment, a composition that provides for the extended releaseof insulin is produced when iminobiotin or iminobiocytin lipidconstructs are loaded with insulin using streptavidin-agarose beads.When the pH of the forementioned construct is adjusted from pH 9.5 to pH4.5 insulin will precipitate within the lipid construct at approximatelypH 5.9. The isoelectric point of insulin is at pH 5.9 and represents thepH at which insulin has its lowest water-solubility. Over a pH rangefrom pH 5.9 to pH 6.7 insulin remains essentially insoluble and exhibitsproperties that are commonly attributed to particulate matter. Theinsolubilized insulin within a lipid construct creates a novel insulinformulation that provides for the time-release of insulin molecules whenadministered by subcutaneous injection or through oral dosing.Solubilization of insulin is initiated as the pH of the lipid constructapproaches pH 7.4.

The lipid construct is freeze-dried or kept in a non-aqueous environmentprior to dosing. In an aqueous dosage form of insulin, the pH of theinsulin solution is maintained at approximately pH 6.5 in order tomaintain insulin in the insoluble form. When insulin is exposed to anexternal pH gradient in vivo insulin is solubilized and move from thelipid construct, thereby supplying insulin to other virus-harboringtissues. Insulin remaining with the lipid construct maintains thecapability of being directed to the hepatocyte binding receptor on thehepatocytes in the liver. Therefore two forms of insulin are producedfrom this particular lipid construct. In an in vivo setting, free andlipid associated insulin are generated in a time-dependent manner. It isanticipated that the solubilization of insulin that is lipid associated,as previously described, can be manufactured to release of insulin overa designated time-release period. This could lead to less frequentdosing schedules for patients afflicted with diabetes.

In a preferred embodiment, insulin molecules move into the lipidconstruct and become sequestered within the lipid domains of the loadedlipid construct. A vector-driven process is employed to move insulinmolecules in one direction during the final phase of the insulin loadingprocedure when the chemical equilibrium is disrupted. During the finalphase of insulin loading, the buffer or aqueous media is rapidly removedso that the insulin molecules associated with the lipid construct aredeprived of an external media into which to migrate. Removal of theexternal media effectively quenches the equilibrium between insulinassociated with the lipid construct and insulin solubilized in theexternal media. This process is termed non-equilibrium loading, asdescribed elsewhere herein.

In an embodiment, a lipid construct is loaded with insulin usingequilibrium methods, an insulin concentration of 273,000 units ofinsulin per microgram of protein is selected to initiate the loadingprocedure. Equilibrium loading continues until the lipid construct issaturated with insulin.

The end process of non-equilibrium loading of insulin into the lipidconstruct requires using a procedure that separates the solid lipidconstruct from the buffered media containing free insulin. In anembodiment, a filtration procedure with a very fine micro-pore syntheticmembrane is used to separate the lipid construct from the externalmedia. In another embodiment, a centricon device equipped with anappropriate filter with a 100,000 molecular weight cut off membrane,such as NanoSep filter is used to remove the lipid construct from thebuffered media containing free insulin. The concentration of insulin inthe lipid construct is maintained because associated insulin is nolonger in equilibrium with the free insulin molecules located in thebulk phase media that had been removed from the construct. Free insulinwhich was in solution is available to load other lipid constructs. Thus,the vector-driven process of concentrating insulin within the lipidconstruct is achieved in one-step in essentially a time-independentprocedure.

After the lipid construct is isolated from the bulk phase media, it canrange in size from approximately 0.0200 microns to 0.4000 microns indiameter. Lipid constructs comprise different particle sizes thatgenerally follow a Gaussian distribution. The appropriate size of thelipid construct needed to achieve the intended pharmacological efficacycan be selected from lipid constructs that comprise particle sizes in aGaussian distribution by the hepatocyte binding receptor.

The lipid construct comprising insulin, lipids and the hepatocytereceptor binding molecule is prepared by using a micro-fluidizationprocess that provides a high shear force which degrades larger lipidconstructs into smaller constructs. The amphipathic lipid constituentsof the lipid construct are 1,2-distearoyl-sn-glycero-3-phosphocholine,cholesterol, dicetyl phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt),triethylammonium 2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate and appropriate derivatives thereof whoserepresentative structures are depicted in Table 1.

In an embodiment, a construct comprises a target molecule complexcomprising multiple linked individual units formed by complexing abridging component with a complexing agent. Typically the targetmolecule complex is formed by combining the selected metal compound, e.g. chromium chloride (III) hexahydrate, with an aqueous bufferedsolution of the complexing agent. In an embodiment, an aqueous bufferedsolution of the complexing agent is prepared by dissolving thecomplexing agent, e.g., N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetic acid, in an aqueous buffered solution, e.g., 10 mMsodium acetate buffer at a final pH of 3.2-3.3. The metal compound isadded in excess in an amount sufficient to complex with an isolatableportion of the complexing agent, and the reaction is conducted at atemperature of 20° C. to 33° C. for 24 to 96 hours, or until theresultant complex precipitates out of aqueous buffered solution. Theprecipitated complexing agent, which demonstrates polymeric properties,is then isolated for future use. This complex is added to the mixture ofamphipathic lipid molecules and an extended amphipathic lipid prior topreparing a lipid construct.

Methods of manufacturing a composition of an insulin in which theisoelectric point was altered by changing the amino acid sequence can beincorporated into a water insoluble target molecule complex are givenbelow. In an embodiment, glargine insulin is incorporated into a waterinsoluble target molecule complex. FIG. 15 demonstrates an outline for aprocess for manufacturing a mixture of free glargine insulin andglargine insulin associated with a water insoluble target moleculecomplex. In an embodiment, the manufacture of the composition involvesthree overall steps: preparing a target molecule complex, incorporatingthe target molecule complex into a lipid construct, and combining thetarget molecule complex with glargine insulin to form a pharmaceuticalcomposition.

The target molecule complex comprises multiple individual units linkedtogether in a polymeric array. Each unit comprises a bridging componentand a complexing agent. In an embodiment, the target molecule complex isformed by combining the selected metal compound, e. g. chromium chloride(III) hexahydrate, with an aqueous buffered solution of the complexingagent. In an embodiment, an aqueous buffered solution of the complexingagent is prepared by dissolving a complexing agent, e.g.,N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid, in anaqueous buffered solution, e.g., 10 mM sodium acetate buffer at a finalpH of 3.2-3.3. A metal compound is added in excess in an amountsufficient to complex with an isolatable portion of the complexingagent, and the reaction is conducted at a temperature of approximately20° C. to 33° C. for approximately 24 to 96 hours, or until theresultant complex precipitates out of the aqueous buffered solution. Theprecipitated complex is then isolated for future use.

The precipitated complex is then mixed with the selected lipids or thelipids of the lipid construct and dissolved in an organic solvent. In anembodiment, the organic solvent is chloroform:methanol (2:1 v/v). Thelipids are in a concentration sufficient to dissolve and incorporateeither all or a portion of the metal complex therein. The mixture of thecomplex and the selected lipids that form the lipid construct aremaintained at a temperature of approximately 60° C. when a hightransition temperature lipid, such as1,2-distearoyl-sn-glycero-3-phosphocholine, is employed. Lowertemperatures may be used depending upon the transition temperature ofthe lipids selected for incorporation into the lipid construct. A timeperiod from 30 minutes to 2 hours under vacuum is generally required todry the lipids and remove any residual organic solvent from the lipidmatrix in order to form the target molecule complex intermediate.

Lipids are produced and loaded by the methods disclosed herein, andthose methods described in U.S. Pat. Nos. 4,946,787; 4,603,044; and5,104,661, and the references cited therein. Typically, the aqueouslipid construct formulations of the invention will comprise 0.1% to 10%active agent by weight (i.e. 1-100 mg drug per ml), and 0.1% to 4% lipidby weight in an aqueous solution, optionally containing salts andbuffers, in a quantity to make 100% by volume. Preferred areformulations which comprise 0.01% to 5% active agent. Most preferred isa formulation comprising 0.01% to 5% active agent by weight and up to 2%by weight of a lipid component in an amount of aqueous solutionsufficient (q. s.) to make 100% by volume.

In an embodiment, glargine insulin was loaded into the target moleculecomplex after the pH of a suspension of the target molecule complex andWater for Injection, USP was adjusted from approximately pH 4.89±0.2 to5.27±0.5. The pH of a solution of glargine insulin was adjusted from pH3.88±0.2 to approximately pH 4.78±0.5, then the water insoluble targetmolecular complex was added. The resulting composition was a mixture offree glargine insulin and glargine insulin associated with a waterinsoluble target molecule complex. A portion of glargine insulin becameassociated with the lipid construct matrix or entrapped in the corevolume of the lipid construct. This pharmaceutical composition is alsoreferred to as HDV-glargine. In an embodiment, an aliquot of the targetmolecule complex is introduced into a vial of Glargine Insulincontaining 100 International units of insulin/ml to provide a hepatocytespecific delivery system containing both free glargine insulin andglargine insulin associated with the target molecule complex.

A pharmaceutical composition that combines free glargine insulin andglargine insulin associated with a water insoluble target moleculecomplex was prepared by the following procedure. The pH of a sample ofSterile Water for Injection, USP, was adjusted to pH 3.95±0.2. Analiquot of HDV suspension was taken and its pH was adjusted in a seriesof steps until the final pH was 5.2±0.5. An aliquot of the Sterile Waterfor Injection, USP, at pH 3.95±0.2 was mixed with the suspension of thetarget molecule complex. The pH of the resulting suspension was4.89±0.2. The pH of this suspension was then adjusted to pH 5.27±0.5.The pH of an aliquot of glargine insulin was adjusted from pH 3.88±0.2to pH 4.78±0.5. This solution was then added to the suspension of thetarget molecule complex at pH 5.20±0.5. The resulting pharmaceuticalcomposition is a mixture of free glargine insulin and glargine insulinassociated with a water insoluble target molecule complex. Thispharmaceutical composition is also referred to as HDV-glargine.

Methods of manufacturing a composition of an insulin in which theisoelectric point was altered by binding charged organic molecules toinsulin can be incorporated into a water insoluble target moleculecomplex are given below. In an embodiment, recombinant human insulinisophane is incorporated into a water insoluble target molecule complex.FIG. 16 demonstrates an outline for a process for manufacturing amixture of free recombinant human insulin isophane, free recombinanthuman regular insulin and a mixture of recombinant human insulinisophane and recombinant human regular insulin that are associated witha water insoluble target molecule complex. In an embodiment, themanufacture of the composition involves three overall steps: preparing atarget molecule complex, incorporating the target molecule complex intoa lipid construct that contains free and associated recombinant humanregular insulin, and combining the target molecule complex with free andassociated recombinant human insulin isophane to form a pharmaceuticalcomposition.

The target molecule complex comprises multiple individual units linkedtogether in a polymeric array. Each unit comprises a bridging componentand a complexing agent. In an embodiment, the target molecule complex isformed by combining the selected metal compound, e. g. chromium chloride(III) hexahydrate, with an aqueous buffered solution of the complexingagent. In an embodiment, an aqueous buffered solution of the complexingagent is prepared by dissolving a complexing agent, e.g.,N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid, in anaqueous buffered solution, e.g., 10 mM sodium acetate buffer at a finalpH of 3.2-3.3. A metal compound is added in excess in an amountsufficient to complex with an isolatable portion of the complexingagent, and the reaction is conducted at a temperature of approximately20° C. to 33° C. for approximately 24 to 96 hours, or until theresultant complex precipitates out of the aqueous buffered solution. Theprecipitated complex is then isolated for future use.

The precipitated complex is then mixed with the selected lipids or thelipids of the lipid construct and dissolved in an organic solvent. In anembodiment, the organic solvent is chloroform:methanol (2:1 v/v). Thelipids are in a concentration sufficient to dissolve and incorporateeither all or a portion of the metal complex therein. The mixture of thecomplex and the selected lipids that form the lipid construct aremaintained at a temperature of approximately 60° C. when a hightransition temperature lipid, such as1,2-distearoyl-sn-glycero-3-phosphocholine, is employed. Lowertemperatures may be used depending upon the transition temperature ofthe lipids selected for incorporation into the lipid construct. A timeperiod from 30 minutes to 2 hours under vacuum is generally required todry the lipids and remove any residual organic solvent from the lipidmatrix in order to form the target molecule complex intermediate.

Lipids can be produced and loaded by the methods disclosed herein, andthose methods described in U.S. Pat. Nos. 4,946,787; 4,603,044; and5,104,661, and the references cited therein. Typically, the aqueouslipid construct formulations of the invention will comprise 0.1% to 10%active agent by weight (i.e. 1-100 mg drug per ml), and 0.1% to 4% lipidby weight in an aqueous solution, optionally containing salts andbuffers, in a quantity to make 100% by volume. Preferred areformulations which comprise 0.01% to 5% active agent. Most preferred isa formulation comprising 0.01% to 5% active agent by weight and up to 2%by weight of a lipid component in an amount of aqueous solutionsufficient (q. s.) to make 100% by volume.

In an embodiment, Humulin NPH insulin was added to a previously formedmixture of recombinant human regular insulin and a lipid construct. Theresulting composition was a mixture of free recombinant human regularinsulin and free recombinant human insulin isophane. Likewise a portionof recombinant human regular insulin and recombinant human insulinisophane is associated with the lipid construct matrix or entrapped inthe core volume of the lipid construct. This pharmaceutical compositionis also referred to as HDV-NPH insulin. In an embodiment, an aliquot ofthe target molecule complex is introduced into a vial of recombinanthuman insulin isophane to provide a hepatocyte specific delivery systemcontaining both free recombinant human insulin isophane and recombinanthuman insulin isophane associated with the target molecule complex. Inan embodiment, recombinant human insulin isophane can be combined withother forms of insulin such as the rapid acting Humalog insulin andNovolog insulin, short acting Regular® insulin, intermediate actingLente insulin and long acting Ultralente insulin and Lantus insulin, orpremixed combinations of insulin. An aliquot of recombinant humaninsulin isophane can be added to a mixture of the target moleculecomplex combined with an insulin that is not recombinant human insulinisophane.

Description of the Invention—Method of Use

Patients with Type I or Type II diabetes are administered an effectiveamount of a hepatocyte targeted lipid construct comprising anamphipathic lipid, an extended amphipathic lipid and insulin. When thiscomposition is administered subcutaneously, a portion of the compositionenters the circulatory system where the composition is transported tothe liver and other areas where the extended amphipathic lipid binds thelipid construct to receptors of hepatocytes. A portion of theadministered composition is exposed to an external gradient in vivowhere insulin can be solubilized and then move from the lipid constructthereby supplying insulin to the muscle and adipose tissue. Insulin thatremains with the lipid construct maintains the capability of beingdirected to the hepatocyte binding receptor on the hepatocytes in theliver. Therefore two forms of insulin are produced from this particularlipid construct. In an in vivo setting, free and lipid associatedinsulin are generated in a time-dependent manner.

The lipid construct structure of the invention provides a useful agentfor pharmaceutical application for administering insulin to a host.Accordingly, the structures of the invention are useful aspharmaceutical compositions in combination with pharmaceuticallyacceptable carriers. Administration of the structures described hereincan be via any of the accepted modes of administration for insulin thatare desired to be administered. These methods include oral, parenteral,nasal and other systemic or aerosol forms.

Oral administration of a pharmaceutical composition comprising insulinassociated with a target molecule complex is followed by intestinalabsorption of insulin associated with the target molecule complex intothe circulatory system of the body where it is also exposed to thephysiological pH of the blood. The lipid construct is targeted fordelivery to the liver. In an embodiment, the lipid construct is shieldedby the presence of cellulose acetate hydrogen phthalate within theconstruct. In the case of oral administration, the shielded lipidconstruct transverses the oral cavity, migrates through the stomach andmoves into the small intestine where the alkaline pH of the smallintestine degrades the cellulose acetate hydrogen phthalate shield. Thede-shielded lipid construct is absorbed into the circulatory system.This enables the lipid construct to be delivered to the sinusoids of theliver. A receptor binding molecule, such as1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl) orother forementioned hepatocyte specific molecules, provides a means forlipid construct to bind to the receptor and then be engulfed orendocytosed by the hepatocytes. Insulin is then released from the lipidconstruct where, upon gaining access to the cellular environment, itperforms its designated function with regard to acting as an agent tocontrol diabetes.

The amount of insulin administered will be dependent on the subjectbeing treated, the type and severity of the affliction, the manner ofadministration and the judgment of the prescribing physician. Althougheffective dosage ranges for specific biologically active substances ofinterest are dependent upon a variety of factors, and are generallyknown to one of ordinary skill in the art, some dosage guidelines can begenerally defined. For most forms of administration, the lipid componentwill be suspended in an aqueous solution and generally not exceed 4.0%(w/v) of the total formulation. The drug component of the formulationwill most likely be less than 20% (w/v) of the formulation and generallygreater than 0.01% (w/v).

Methods of administering a composition of an insulin in which theisoelectric point was altered by changing the amino acid sequence isincorporated into a water insoluble target molecule complex are givenbelow. In an embodiment, patients with Type I or Type II diabetes areadministered an effective amount of a hepatocyte targeted compositioncomprising a mixture of free glargine insulin and glargine insulinassociated with a water insoluble target molecule complex. In anembodiment, glargine insulin can be combined with other forms ofinsulin, such as insulin lispro, insulin aspart, regular insulin,insulin zinc, human insulin zinc extended, isophane insulin, humanbuffered regular insulin, insulin glulisine, recombinant human regularinsulin, recombinant human insulin isophane or premixed combinations ofany of the aforementioned insulins, a derivative thereof, and acombination of any of the aforementioned insulins. In an embodiment, thecomposition can be administered by a subcutaneous or oral route.

The lipid construct structure of the invention provides a useful agentfor pharmaceutical application for administering insulin to a host.Accordingly, the structures of the invention are useful aspharmaceutical compositions in combination with pharmaceuticallyacceptable carriers. Administration of the structures described hereincan be via any of the accepted modes of administration for insulin thatare desired to be administered. These methods include oral, parenteral,nasal and other systemic or aerosol forms.

After a composition is administered to a patient by subcutaneousinjection, the in situ physiological environment in the injection area,the morphology and chemical structures of free glargine insulin and theglargine insulin associated with the water insoluble target moleculecomplex begin to change. As the pH of the environment around the freeglargine insulin and the glargine insulin associated with the waterinsoluble target molecule complex increases after being diluted withphysiological media, the pH reaches the isoelectric point of glargineinsulin, where flocculation, aggregation and precipitation reactionsoccur for both free glargine insulin and glargine insulin associatedwith the target molecule complex. The rates at which these processesoccur differ between free glargine insulin and glargine insulinassociated with the target molecule complex. The free glargine insulinis directly exposed to changes in pH and dilution. Exposure of glargineinsulin associated with the target molecule complex to small changes inpH and dilution at physiological pH is delayed due to the time requiredfor diffusion of physiological fluids or media through the lipid bilayerin the water insoluble target molecule complex. The delay in the releaseof insulin from the lipid construct as well as the delay of the releaseof lipid construct with associated insulin within the precipitated freeglargine matrix is an essential feature of the invention since itaffects and augments the biological and pharmacological response invivo.

Oral administration of a pharmaceutical composition that combines freeglargine insulin and glargine insulin associated with a target moleculecomplex is followed by intestinal absorption of glargine insulinassociated with the target molecule complex into the circulatory systemof the body where it is also exposed to the physiological pH of theblood. All or a portion of the lipid construct is delivered to theliver.

As the physiological dilution is increased in situ in the subcutaneousspace or upon entering into the circulatory system, the free glargineinsulin and glargine insulin associated with the target molecule complexencounter a normal physiological pH environment of pH 7.4. As a result,free glargine insulin changes from a soluble form at injection, to ainsoluble form at a pH near its isoelectric point of pH 5.8-6.2, andthen to a soluble form at physiological pH. In the soluble form,glargine insulin migrates through the body to sites where it is capableof eliciting a pharmacological response. Glargine insulin associatedwith the water insoluble target molecule complex becomes solubilized andreleased from the complex at a different rate that is slower than thatof free glargine insulin. This is because glargine insulin associatedwith the water insoluble target molecule complex has to traverse thecore volume and lipid domains of the water insoluble target moleculecomplex before it contacts the bulk phase media.

The amount of glargine insulin administered will be dependent on thesubject being treated, the type and severity of the affliction, themanner of administration and the judgment of the prescribing physician.Although effective dosage ranges for specific biologically activesubstances of interest are dependent upon a variety of factors, and aregenerally known to one of ordinary skill in the art, some dosageguidelines can be generally defined. For most forms of administration,the lipid component will be suspended in an aqueous solution andgenerally not exceed 4.0% (w/v) of the total formulation. The drugcomponent of the formulation will most likely be less than 20% (w/v) ofthe formulation and generally greater than 0.01% (w/v).

Methods of administering a composition of an insulin in which theisoelectric point was altered by binding charged organic molecules toinsulin is incorporated into a water insoluble target molecule complexare given below. In an embodiment, patients with Type I or Type IIdiabetes are administered an effective amount of a hepatocyte targetedcomposition comprising a mixture of free recombinant human insulinisophane plus free recombinant human regular insulin along withrecombinant human insulin isophane and recombinant human regular insulinwhich are both are associated with a water insoluble target moleculecomplex. In an embodiment, recombinant human insulin isophane can becombined with other forms of insulin, such as of insulin lispro, insulinaspart, regular insulin, insulin glargine, insulin zinc, human insulinzinc extended, isophane insulin, human buffered regular insulin, insulinglulisine, recombinant human regular insulin, recombinant human insulinisophane or premixed combinations of any of the aforementioned insulins,a derivative thereof, and a combination of any of the aforementionedinsulins.

The lipid construct structures of the invention provides a useful agentfor pharmaceutical application for administering insulin to a host.Accordingly, the structures of the invention are useful aspharmaceutical compositions in combination with pharmaceuticallyacceptable carriers. Administration of the structures described hereincan be via any of the accepted modes of administration for insulin thatare desired to be administered. These methods include oral, parenteral,nasal and other systemic or aerosol forms.

After a composition is administered to a patient by subcutaneousinjection, the in situ physiological environment in the injection area,the morphology and chemical structures of free recombinant human insulinisophane and the recombinant human insulin isophane associated with thewater insoluble target molecule complex begins to change. As the pH ofthe environment around the free recombinant human insulin isophane andthe recombinant human insulin isophane associated with the waterinsoluble target molecule complex becomes diluted with physiologicalmedia, some solubilization occurs for both insulins. As a result ofsolubilization and equilibrium conditions recombinant human insulinisophane can become associated with the target molecule complex. Therates at which these equilibrium processes occur differ between freerecombinant human insulin isophane and recombinant human insulinisophane associated with the target molecule complex. The freerecombinant human insulin isophane is directly exposed to small changesin pH and physiological dilution. Exposure of recombinant human insulinisophane associated with the target molecule complex to small changes inpH and dilution at physiological pH is delayed due to the time requiredfor diffusion of physiological fluids or media through the lipid bilayerin the water insoluble target molecule complex. The delay in the releaseof insulin from the lipid construct as well as the delay of the releaseof the lipid construct as it exists within the precipitated freerecombinant human insulin isophane matrix is an essential discovery ofthe invention since it affects and augments the biological andpharmacological response in vivo.

Oral administration of a pharmaceutical composition that combines freerecombinant human insulin isophane and recombinant human insulinisophane associated with a target molecule complex is followed byintestinal absorption of recombinant human insulin isophane associatedwith the target molecule complex into the circulatory system of the bodywhere it is also exposed to the physiological pH of the blood. All or aportion of the lipid construct is delivered to the liver.

As the physiological dilution is increased in situ in the subcutaneousspace or upon entering into the circulatory system, free recombinanthuman insulin isophane and recombinant human insulin isophane associatedwith the target molecule complex encounter a normal physiological pHenvironment of pH 7.4. As a result of dilution free recombinant humaninsulin isophane changes from an insoluble form at injection, to asoluble form at physiological pH. In the soluble form, recombinant humaninsulin isophane migrates through the body to sites where it is capableof eliciting a pharmacological response. Recombinant human insulinisophane associated with the water insoluble target molecule complexbecomes solubilized and released from the complex at a different ratethat is slower than that of free recombinant human insulin isophane.This is because recombinant human insulin isophane associated with thewater insoluble target molecule complex has to traverse the core volumeand lipid domains of the water insoluble target molecule complex beforeit contacts the bulk phase media.

The lipid construct structure of the invention provides a useful agentfor pharmaceutical application for administering recombinant humaninsulin isophane to a host. Accordingly, the structures of the inventionare useful as pharmaceutical compositions in combination withpharmaceutically acceptable carriers. Administration of the structuresdescribed herein can be via any of the accepted modes of administrationfor recombinant human insulin isophane that are desired to beadministered. These methods include oral, parenteral, nasal and othersystemic or aerosol forms.

The amount of recombinant human insulin isophane and recombinant humanregular insulin administered will be dependent on the subject beingtreated, the type and severity of the affliction, the manner ofadministration and the judgment of the prescribing physician. Althougheffective dosage ranges for specific biologically active substances ofinterest are dependent upon a variety of factors, and are generallyknown to one of ordinary skill in the art, some dosage guidelines can begenerally defined. For most forms of administration, the lipid componentwill be suspended in an aqueous solution and generally not exceed 4.0%(w/v) of the total formulation. The drug component of the formulationwill most likely be less than 20% (w/v) of the formulation and generallygreater than 0.01% (w/v).

The amount of insulin administered will be dependent on the subjectbeing treated, the type and severity of the affliction, the manner ofadministration and the judgment of the prescribing physician. Althougheffective dosage ranges for specific biologically active substances ofinterest are dependent upon a variety of factors, and are generallyknown to one of ordinary skill in the art, some dosage guidelines can begenerally defined. For most forms of administration, the lipid componentwill be suspended in an aqueous solution and generally not exceed 4.0%(w/v) of the total formulation. The drug component of the formulationwill most likely be less than 20% (w/v) of the formulation and generallygreater than 0.01% (w/v).

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 5% with the balance made up from non-toxic carriers may beprepared.

The exact composition of these formulations may vary widely depending onthe particular properties of the drug in question. However, they willgenerally comprise from 0.01% to 5%, and preferably from 0.05% to 1%active ingredient for highly potent drugs, and from 2%-4% for moderatelyactive drugs.

The percentage of active ingredient contained in such parenteralcompositions is highly dependent on the specific nature thereof, as wellas the activity of the active ingredient and the needs of the subject.However, percentages of active ingredient of 0.01% to 5% in solution areemployable, and will be higher if the composition is a solid which willbe subsequently diluted to the above percentages. Preferably thecomposition will comprise 0.2%-2.0% of the active agent in solution.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other ingredients, and then, if necessary or desirable, shaping orpackaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, parenteral, pulmonary, intranasal, buccal, or another route ofadministration.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage. However, delivery ofthe active agent as set forth in the invention may be as low as 1/10,1/100 or 1/1,000 or smaller than the dose normally administered becauseof the targeted nature of the insulin therapeutic agent.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and U.S. Pat. No. 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, kaolin or celluloseacetate hydrogen phthalate.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampoules or in multi-dosecontainers containing a preservative. Formulations for parenteraladministration include, but are not limited to, suspensions, solutions,emulsions in oily or aqueous vehicles, pastes, and implantablesustained-release or biodegradable formulations. Such formulations mayfurther comprise one or more additional ingredients including, but notlimited to, suspending, stabilizing, or dispersing agents. In oneembodiment of a formulation for parenteral administration, the activeingredient is provided in dry (i.e. powder or granular) form forreconstitution with a suitable vehicle (e.g. sterile pyrogen-free water)prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a lipid construct preparation, or as acomponent of a biodegradable polymer system. Compositions for sustainedrelease or implantation may comprise pharmaceutically acceptablepolymeric or hydrophobic materials such as an emulsion, an ion exchangeresin, a sparingly soluble polymer, or a sparingly soluble salt.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 microns, and preferably from about 1 to about6 microns. Such compositions are conveniently in the form of dry powdersfor administration using a device comprising a dry powder reservoir towhich a stream of propellant may be directed to disperse the powder orusing a self-propelling solvent/powder-dispensing container such as adevice comprising the active ingredient dissolved or suspended in alow-boiling propellant in a sealed container. Preferably, such powderscomprise particles wherein at least 98% of the particles by weight havea diameter greater than 0.5 microns and at least 95% of the particles bynumber have a diameter less than 7 microns. More preferably, at least95% of the particles by weight have a diameter greater than 1 nanometerand at least 90% of the particles by number have a diameter less than 6microns. Dry powder compositions preferably include a solid fine powderdiluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 microns.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 microns. Such a formulation is administered in themanner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 75% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 microns, and may further comprise one or more ofthe additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1%-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a lipid construct preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically dosages of the active ingredient in the composition of theinvention which may be administered to an animal, preferably a human,range in amount from 1 micrograms to about 100 g per kilogram of bodyweight of the animal. While the precise dosage administered will varydepending upon any number of factors, including but not limited to, thetype of animal and type of disease state being treated, the age of theanimal and the route of administration. Preferably, the dosage of theactive ingredient will vary from about 1 mg to about 10 g per kilogramof body weight of the animal. More preferably, the dosage will vary fromabout 10 mg to about 1 g per kilogram of body weight of the animal.

The composition may be administered to an animal as frequently asseveral times daily, or it may be administered less frequently, such asonce a day, once a week, once every two weeks, once a month, or evenlees frequently, such as once every several months or even once a yearor less. The frequency of the dose will be readily apparent to theskilled physician and will depend upon any number of factors, such as,but not limited to, the type and severity of the disease being treated,the type and age of the animal, etc.

The invention also includes a kit comprising the composition of theinvention and an instructional material which describes administeringthe composition to a tissue of a mammal. In another embodiment, this kitcomprises a (preferably sterile) solvent suitable for dissolving orsuspending the composition of the invention prior to administering thecomposition to the mammal.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the protein of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviation the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the components of the invention or be shipped togetherwith a container which contains the components of the invention.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the instructional material and thecomposition be used cooperatively by the recipient.

The pharmaceutical compositions useful for practicing the invention maybe administered to deliver a dose equivalent to standard doses ofinsulin.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, companion animals and othermammals.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral or injectable routes of administration.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

The materials and methods used in the experiments presented in thisExperimental Example are now described.

Experimental Example 1. Pharmaceutical Composition 1

A lipid construct comprises a mixture of the lipids1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetylphosphate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt),the receptor binding molecule1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl) andinsulin.

Experimental Example 2. Pharmaceutical Composition 2

A lipid construct comprises a mixture of the lipids1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetylphosphate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycero)] (sodium salt),insulin, the receptor binding molecule1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl),and/or polychromium-poly(bis)-[N-(2,6-(diisopropylphenyl)carbamoylmethyl) imino]diacetic acid]. The lipid anchoring-hepatocytereceptor binding molecule1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl) andpolychromium-poly(bis)-[N-(2,6-(diisopropylphenyl)carbamoyl methyl)iminodiacetic acid] had been added to the lipid construct at a level of1.68%±0.5% by weight and 1.2%±0.5% by weight, respectively.

Experimental Example 3. Pharmaceutical Composition 3

A lipid construct comprises a mixture of the amphipathic lipids1,2-distearoyl-sn-glycero-3-phosphocholine (12.09 g), cholesterol (1.60g), dicetyl phosphate (3.10 g),polychromium-poly(bis)-[N-(2,6-(diisopropylphenyl)carbamoylmethyl)imino] diacetic acid] (0.20 g) and insulin. The mixturewas added to a aqueous medium and the total mass was 1200 g.

Experimental Example 4. Preparation of a Lipid Construct ContainingInsulin

The lipid construct was formed by preparing a mixture of amphipathiclipid molecules and an extended amphipathic lipid, preparing a lipidconstruct from the mixture of amphipathic lipid molecules and anextended amphipathic lipid, and combining insulin into the lipidconstruct.

A mixture of amphipathic lipid molecules and an extended amphipathiclipid was produced using the following procedure. A mixture of the lipidcomponents [total mass of 8.5316 g] of the lipid construct was preparedby combining aliquots of the lipids1,2-distearoyl-sn-glycero-3-phosphocholine (5.6881 g), cholesterolcrystalline (0.7980 g), dicetyl phosphate (1.5444 g),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl)(0.1436 g), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (0.1144 g),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (0.1245 g)and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodiumsalt) (0.1186 g).

A 100 ml solution of chloroform:methanol (2:1 v:v) was dehydrated over5.0 grams of molecular sieves. The mixture of the lipid components oflipid construct was placed in a 3 liter flask and 45 mls of thechloroform/methanol solution was added to the lipid mixture. Thesolution was placed in flask on a rotoevaporator with a water bath at60° C.±2° C. and turned slowly. The chloroform/methanol solution wasremoved under vacuum on a rotary evaporator using an aspirator forapproximately 45 minutes, followed by a vacuum pump for approximatelytwo hours to remove residual solvent, and the solid mixture of thelipids formed. The dried mixture of lipids can be stored in a freezer atapproximately −20° C.-0° C. for an indefinite time period.

The lipid construct was prepared from the mixture of amphipathic lipidmolecules and an extended amphipathic lipid using the followingprocedure. The lipid mixture was mixed with approximately 600 ml of 28.4mM sodium phosphate (monobasic-dibasic) buffer at pH 7.0. The lipidmixture was swirled, then placed in a heated water bath at 80° C.±4° C.for 30 minutes while slowly turning to hydrate the lipids.

A M-110 EHI microfluidizer was preheated to 70° C.±10° C. using SWI witha pH between 6.5-7.5. The suspension of the hydrated target complex wastransferred to the microfluidizer and microfluidized at approximately9000 psig using one pass of the suspension of the hydrated targetmolecule complex through the fluidizer. After passing through themicrofluidizer, an unfiltered sample (2.0-5.0 ml) of the fluidizedsuspension was collected for particle size analysis using unimodaldistribution data from a Coulter N-4 plus particle size analyzer. Priorto all particle size determinations, the sample was diluted with 0.2micron filtered SWI that has been pH adjusted to between 6.5-7.5. Theparticle size was required to range from 0.020-0.40 microns. If theparticle size was not within this range, the suspension was passedthrough the microfluidizer again at approximately 9000 psig, and theparticle size was analyzed again until the particle size requirementsare reached. The microfluidized target molecule complex was collected ina sterile container.

The microfluidized target molecule complex was maintained at 60° C.±2°C. while filtered twice through a sterile 0.8 micron+0.2 micron gangfilter attached to a 5.0 ml syringe. An aliquot of the filteredsuspension was analyzed to determine the particle size range ofparticles in the suspension. The particle size range of the final 0.2micron filtered sample should be in the range from 0.0200-0.2000 micronsas determined from the unimodal distribution printout from the particlesize analyzer.

Insulin is loaded into the construct by reverse loading of the constructusing the methods described in U.S. Pat. No. 5,104,661, which isincorporated herein by reference.

Experimental Example 5. Method of Use

The efficacy of hepatic directed vesicle (HDV) insulin on hepaticglycogen was evaluated in a rat model. A total of 60 Male Sprague-Dawleyrats (8 weeks of age; 250 g) were divided into five treatment groups asdescribed below.

For the first day of the study, all rats were fasted for 24 hours withab libitum water. On the second day, the rats were injectedintraperitoneally with a mixture of alloxan and streptozotocin (AS). Themixture of alloxan and streptozotocin was prepared in pH 7 0.01 Mphosphate buffer by weighing 5 mg per mL of each material so that thefinal concentration is 5 mg alloxan per mL and 5 mg streptozotocin permL. The AS mixture was administered 0.5 mL of the mixture of alloxan andstreptozotocin via intraperitoneal injection at 20 mg/kg body weight (10mg/kg alloxan and 10 mg/kg streptozotocin). AS will cause a massiverelease of insulin resulting in a profound and transient hypoglycemia afew hours after injecting AS. A 10% glucose in water solution wasinjected subcutaneously as needed to prevent hypoglycemia and keep therats adequately hydrated during the second day. A normal chow diet andwater were available ad libitum.

On the third day, a baseline tail-vein blood glucose sample is taken at0 Minutes, followed immediately by a subcutaneous injection of one ofthe following solutions at 0.32 U insulin/rat, corresponding to thegroup to which the rat was assigned.

-   (1) HDV-insulin with a Cr-disofenin    [polychromium-poly(bis)-[N-(2,6-(diisopropylphenyl) carbamoyl    methyl)imino diacetic acid]] hepatocyte target molecule (HTM)    (Positive) control. There was no extended amphipathic lipid present.    The amount of amphipathic lipids present provided a dose of about    14.5 micrograms of amphipathic lipids per kilogram of rat.-   (2) Regular insulin (negative) control;-   (3) HDV-insulin test material 1, where the extended amphipathic    lipid was Biotin-X DHPE [triethylammonium 2,3-diacetoxypropyl    2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)    pentanamido)hexanamido)ethyl phosphate]. The amount of amphipathic    lipids present provided a dose of about 14.5 micrograms of    amphipathic lipids per kilogram of rat. The amount of extended    amphipathic lipid present provided a dose of about 191 nanograms of    extended amphipathic lipid per kilogram of rat.-   (4) HDV-insulin test material 2, where the extended amphipathic    lipid was Biotin DHPE [triethylammonium 2,3-diacetoxypropyl    2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)    pentanamido)ethyl phosphate]. The amount of amphipathic lipids    present provided a dose of about 7.25 micrograms of amphipathic    lipids per kilogram of rat. The amount of extended amphipathic lipid    present provided a dose of about 95.5 nanograms of extended    amphipathic lipid per kilogram of rat.-   (5) HDV-insulin test material 3, where the extended amphipathic    lipid was Biotin DHPE [triethylammonium 2,3-diacetoxypropyl    2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)    pentanamido)ethyl phosphate]. The amount of amphipathic lipids    present provided a dose of about 14.5 micrograms of amphipathic    lipids per kilogram of rat. The amount of extended amphipathic lipid    present provided a dose of about 191 nanograms of extended    amphipathic lipid per kilogram of rat.    For treatment groups 1 and 3-5, the amphipathic lipids were a    mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol,    and dicetyl phosphate.

At “0” minutes, each rat was also gavaged with 375 mg glucose in 3.75 mlwater (10% glucose).

Half of the animals of each group were anesthetized and euthanized usingketamine (150 mg/kg)/xylazine (15 mg/kg) at one hour minutes and theremaining rats at 2 hours via I.P. Previous studies with Cr-disofeninHTM have shown the statistically significant effect over 2 hours. Theentire liver was removed and stored in liquid nitrogen at −80° C. untilanalyzed for hepatic glycogen.

Hepatic glycogen was determined by the following procedure which isdescribed by Ong K C and Kho H E, Life Sciences 67 (2000) 1695-1705.Weighed amounts (0.3-0.5 g) of frozen liver tissue were homogenized in10 volumes of ice-cold 30% KOH and then boiled at 100° C. for 30minutes. Glycogen was precipitated with ethanol, pelleted, washed, andresolubilized in distilled water. Glycogen content was determined bytreating the aqueous solution with anthrone reagent (1 g anthronedissolved in 500 ml conc. H₂SO₄). The absorbance of the solution at 625nm was measured in a spectrometer and the amount of glycogen present wascalculated.

The results are shown in FIG. 17, which compares the concentration ofglycogen present in the liver for the five treatment groups. The valuesare the average of the one and two hour values, which were similar toeach other. Regular insulin, which has been shown to be ineffective as astimulant for hepatic glucose and glycogen storage, was used as anegative control. HDV-Insulin with the Cr-disofenin HTM was the positivecontrol and it had a significantly higher glycogen content (p<0.05) thandid the regular insulin negative control. Thus the expected statisticaland biologically significant differences between the negative andpositive controls post dosing were observed.

Test materials 1 and 3, which had the extended amphipathic lipids biotinDHPE [triethylammonium 2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate] and biotin-X DHPE [triethylammonium2,3-diacetoxypropyl2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido) ethyl phosphate] had statistically higher(p=0.05) glycogen levels than did the regular insulin. Test material 2,which also had biotin-X DHPE, but with lipid concentrations one-half ofthose in test material 3, had glycogen levels that were higher, but thewithin group variability was great enough to give a p=0.08.

Experimental Example 6. Pharmaceutical Composition of HDV-GlargineInsulin

A hepatocyte targeted composition comprises a mixture of free glargineinsulin and glargine insulin associated with a water insoluble targetmolecule complex. The complex comprises multiple linked individual unitsand a lipid construct matrix, comprising a mixture of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetylphosphate. The bridging agent polychromium poly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid] is presentwithin the complex.

Experimental Example 7. Preparation of HDV-Glargine Insulin

An intermediate mixture of the components of a target molecule complexwas produced by the following procedure. A mixture of the components[total mass of 2.830 g] of a target molecule complex was prepared byadding aliquots of the lipids 1,2-distearoyl-sn-glycero-3-phosphocholine(2.015 g), crystalline cholesterol (0.266 g), and dicetyl phosphate(0.515 g) to the bridging agent, polychromium poly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid] (0.034 g).A solution of chloroform (50 ml) and methanol (25 ml) had beendehydrated over molecular sieves. The mixture of the components of thetarget molecule complex was added to the chloroform/methanol solution,which was then placed in a water bath at 60° C.±2° C. to form asolution. The chloroform/methanol solution was removed under vacuum on arotary evaporator using an aspirator, followed by a vacuum pump, and thesolid intermediate mixture formed.

A target molecule complex was produced by the following process. The pHof 530 ml of Sterile Water for Injection, USP (SWI) was adjusted tobetween pH 6.5-7.5 by the addition of a 105 μl of 0.1 N NaOH solution.Sufficient water was added to make 200 g of product. The pH adjusted SWIwas added to the intermediate mixture (2.830 g) and the intermediatemixture was hydrated by placing the mixture in a water bath at 80° C.±2°C. while rotating the mixture for approximately 30 minutes±15 minutes,or until the mixture was a uniform appearing suspension. During theprevious process, the pH of the suspension decreased. The pH of thesuspension was then adjusted to pH 5.44±0.5 pH units by the addition ofapproximately 1.0 ml 0.1 N NaOH.

The suspension of the hydrated target complex was transferred to a modelM-110 EHI microfluidizer that was preheated to 70° C.±10° C. with 28 mMsodium phosphate buffer at pH 7.0. The suspension was microfluidized at9,000 psig using one pass of the suspension of the hydrated targetmolecule complex through the fluidizer. After passing through themicrofluidizer, an unfiltered sample (2.0-5.0 ml) of the fluidizedsuspension was collected for particle size analysis using unimodaldistribution data from a Coulter N-4 plus particle size analyzer. Priorto all particle size determinations, the sample was diluted with 0.2micron filtered SWI that has been pH adjusted to between 6.5-7.5. Theparticle size was required to range from 0.020-0.40 microns. If theparticle size was not within this range, the suspension was passedthrough the microfluidizer again, and the particle size was analyzedagain until the particle size requirements was reached. Themicrofluidized target molecule complex was collected in a sterilecontainer.

The suspension of the microfluidized target molecule complex wasmaintained at 60° C.±2° C. while filtered twice through a sterile 0.8micron+0.2 micron gang filter attached to a 5.0 ml syringe. An aliquotof the filtered suspension was analyzed to determine the particle sizerange of particles in the suspension. The particle size of the final 0.2micron filtered sample was in the range from 0.0200-0.2000 microns, asdetermined from the unimodal distribution printout from the particlesize analyzer. The pH of the filtered suspension of the target moleculecomplex was 3.74±0.2 pH units before pH adjustment. Samples were storedin a refrigerator between 2°-8° C. until further use.

The pharmaceutical composition comprising a mixture of free glargineinsulin and glargine insulin associated with a water insoluble targetmolecule complex, also referred to as HDV-glargine insulin, was producedwas produced by the following process. The pH of a 5.0 ml aliquot of thetwice filtered suspension of the target molecule complex was adjustedfrom an initial pH of pH 3.74±0.2 to pH 5.2±pH 0.5 by the sequentialaddition of sterile 0.1 NaOH according to the following procedure:

-   -   pH 3.74+10 μl 0.1 N NaOH→pH 3.96    -   pH 3.96+20 μl 0.1 N NaOH→pH 4.52    -   pH 4.52+10 μl 0.1 N NaOH→pH 4.69    -   pH 4.69+10 μl 0.1 N NaOH→pH 5.01    -   pH 5.01+10 μl 0.1 N NaOH→pH 5.20

A 1.6 ml aliquot of the target molecule complex suspension at pH5.20±0.5 was combined with 18.4 ml of SWI, which had been adjusted to pH3.95±0.2. The pH of the resulting suspension was adjusted from pH 4.89to pH 5.27±0.5 by the addition of 10 μl±1.0 μl of 0.1 N NaOH.

The pH of 5.0 ml aliquot of Lantus® Glargine—U-100 Insulin was increasedfrom pH 3.88±0.2 to pH 4.78±0.5 by the addition of 60 μl±2 μl of sterile0.1 N NaOH with mixing. A 2.5 ml±0.1 ml aliquot of the target moleculecomplex suspension at pH 5.27±0.5 was added to 5.0 ml±0.1 ml of thesolution of Glargine insulin at pH 4.78±0.5 to produce thepharmaceutical composition containing a mixture of free glargine insulinand glargine insulin associated with the water insoluble target moleculecomplex. The product contained 66.1 IU of glargine insulin/mlsuspension. In an embodiment, the mixture of free glargine insulin andglargine insulin associated with the complex can be produced in a vialof glargine insulin in situ in order to manufacture individual dosageforms.

Example 8. Method of Use of HDV-Glargine Insulin for the Control ofBlood Glucose in Type I Diabetes Mellitus Patients

HDV-glargine insulin was administered to patients to determine theability of HDV-glargine insulin to control post prandial blood glucoselevels. Seven Type I diabetes mellitus patients were selected. Thepatients were carefully screened and selected according to criterialisted in the study protocol. The patients were treated with basalglargine insulin and a short-acting insulin at meal times prior toentering the HDV-glargine insulin treatment period. Patients weremonitored (via diary cards and site contact) for four days prior toadministering HDV-glargine insulin to assure that they were inacceptable control of their blood glucose levels. Morning fastingglucose levels were established to be in the range of 100-150 mg/dl.

During the study, the dose of HDV-glargine insulin for each patient was1.2× their usual daily dose of basal glargine insulin to compensate forthe amount of short-acting insulin that they would not receive on thetest days. Blood samples were taken according to a set schedule over 13hours. HDV was added to glargine insulin using the method previouslydescribed to produce a suspension with a final concentration of 66.1 IUglargine/ml and 0.37 mg HDV/ml. The patients were injected withHDV-glargine insulin one hour prior to the morning breakfast. At each ofthe three daily meals, breakfast, lunch and dinner, a 60 gramcarbohydrate meal was prescribed by a dietitian.

The results of the experiments presented in this Experimental Exampleare now described. HDV-glargine insulin was well tolerated by thepatients and no adverse reactions were observed at the injection sites.Hypoglycemic reactions were not observed in patients receiving thistreatment. The blood glucose values of patients treated withHDV-glargine insulin are graphically presented in FIG. 18. FIG. 18 showsthat blood glucose concentrations increased, as anticipated, followingmeals and glucose concentrations decreased over time until the next mealwas eaten. This pattern was observed for all four patients. FIG. 19shows the effect of a single dose of HDV-glargine insulin on averageblood glucose concentrations in patients consuming three meals duringthe day. As with the individual patients, blood glucose concentrationsincreased following meals and glucose concentrations decreased over timeuntil the next meal was eaten. Average blood glucose concentrations wereabove the baseline value at all time points. The curve suggests that theefficacy of HDV-glargine insulin improved throughout the day becausethere was less variation between the high and low concentrations afterthe lunch and dinner meals than the breakfast meal. The effect ofHDV-glargine insulin on blood glucose concentrations over time relativeto blood glucose concentrations during fasting are shown in FIG. 20.Blood glucose concentrations increased following meals then decreasedover time towards the glucose concentration during fasting until thenext meal was eaten. Blood glucose concentrations were above fastingconcentrations throughout the study. Treatment of patients withHDV-glargine insulin resulted in some degree of post-prandial bloodglucose level control, indicating that HDV was able to carry sufficientquantities of glargine-insulin to the liver at mealtimes to provide thiscontrol. Blood glucose levels were typical of Type I patients thatusually receive basal insulin therapy plus short-acting insulins at mealtimes.

Experimental Example 9. Pharmaceutical Composition of HDV-Humulin NPHInsulin #1

A hepatocyte targeted composition comprises a mixture of freerecombinant human insulin isophane and recombinant human insulinisophane associated with a water insoluble target molecule complex. Thecomplex comprises multiple linked individual units and a lipid constructmatrix comprising a mixture of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetylphosphate. The bridging agent polychromiumpoly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid]is present within the complex.

Experimental Example 10. Pharmaceutical Composition of HDV-Humulin NPHInsulin #2

A hepatocyte targeted composition comprises a mixture of freerecombinant human insulin isophane, free recombinant human regularinsulin, and recombinant human insulin isophane and recombinant humanregular insulin associated with a water insoluble target moleculecomplex. The complex comprises multiple linked individual units and alipid construct matrix comprising a mixture of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetylphosphate. The bridging agent polychromiumpoly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid]is present within the complex.

Experimental Example 11. Preparation of HDV-Humulin NPH Insulin

An intermediate mixture of the components of a target molecule complexwas produced by the following procedure. A mixture of the components[total mass of 2.830 g] of a target molecule complex was prepared byadding aliquots of the lipids 1,2-distearoyl-sn-glycero-3-phosphocholine(2.015 g), crystalline cholesterol (0.266 g), and dicetyl phosphate(0.515 g) to the bridging agent, polychromiumpoly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid](0.034 g). A solution of chloroform (50 ml) and methanol (25 ml) hadbeen dehydrated over molecular sieves. The mixture of the components ofthe target molecule complex was added to 25.0 mls thechloroform/methanol solution, which was then placed in a water bath at60° C.±0.2 C to form a solution. The chloroform/methanol solution wasremoved under vacuum on a rotary evaporator using an aspirator, followedby a vacuum pump, and the solid intermediate mixture formed.

A target molecule complex was produced by the following process.Approximately 200 ml of 28 mM sodium phosphate buffer at pH 7.0 wasadded to the intermediate mixture to form a aqueous suspension. Theaqueous suspension was hydrated in a water bath at 80° C.±2° C. whilerotating the mixture for approximately 30 minutes±15 minutes or untilthe mixture was a uniform appearing suspension.

The suspension of the hydrated target complex was transferred to a modelM-110 EHI microfluidizer that was preheated to 70° C.±10° C. with 28 mMsodium phosphate buffer at pH 7.0. The suspension was microfluidized at9,000 psig using one pass of the suspension of the hydrated targetmolecule complex through the fluidizer. After passing through themicrofluidizer, an unfiltered sample (2.0-5.0 ml) of the fluidizedsuspension was collected for particle size analysis using unimodaldistribution data from a Coulter N-4 plus particle size analyzer. Priorto all particle size determinations, the sample was diluted with 28 mMsodium phosphate buffer pH 7.0. If the particle size was not within therange of 0.020-0.40 microns, the suspension was passed through themicrofluidizer again, and the particle size was analyzed again. This isrepeated until the particle size is within the range of 0.020-0.40microns. The suspension of the microfluidized target molecule complexwas collected in a sterile container.

The suspension of the microfluidized target molecule complex wasmaintained at 60° C.±2° C. while filtered through a sterile 0.8micron+0.2 micron gang filter attached to a 5.0 ml syringe. An aliquotof the filtered suspension was analyzed to determine the particle sizerange of particles in the suspension. The particle size of the final 0.2micron filtered sample was in the range from 0.0200-0.2000 microns, asdetermined from the unimodal distribution printout from the particlesize analyzer. The pH of the filtered suspension of the target moleculecomplex was 7.0±0.5 pH units. Samples were stored in a refrigeratorbetween 2°-8° C. until further use.

The filtered HDV-lipid suspension contained 14.15 mg of HDV lipid/ml. A0.8 ml aliquot of this suspension was added to a 10.0 ml vial of HumulinR insulin and allowed to incubate for several days at 2°-8° C. Then 5.0ml of the 10.0 ml Humulin R insulin HDV suspension was removed with asterile syringe. To the remaining 5.0 ml of Humulin R insulin in thevial, 5.0 ml of Humulin NPH insulin was added to form the final HDVproduct. The final HDV composition contained 93.6 units of combined HDVHumulin R and HDV Humulin NPH insulin/ml of suspension and 0.52 mg ofHDV lipid/ml. This composition, which can be produced in situ tomanufacture individual dosage forms, comprised a mixture of free HumulinR insulin, free Humulin NPH insulin and both Humulin R insulin andHumulin NPH insulin associated with a lipid construct.

Example 12. Method of Use of Combined HDV Humulin R Insulin andHDV-Humulin NPH Insulin for the Control of Blood Glucose in Type IDiabetes Mellitus Patients

HDV-Humulin NPH insulin was administered to patients to determine theability of HDV-Humulin NPH insulin to control post prandial bloodglucose levels. Seven Type I diabetes mellitus patients were selected.The patients were carefully screened and selected according to criterialisted in the study protocol. The patients were treated with basalHumulin NPH insulin and a short-acting insulin at meal times prior toentering the HDV-Humulin NPH insulin treatment period. Patients weremonitored (via diary cards and site contact) for four days prior toadministering HDV-Humulin NPH insulin to assure that they were inacceptable control of their blood glucose levels. Morning fastingglucose levels were established to be in the range of 100-150 mg/dl.

During the study, the dose of HDV-Humulin NPH insulin for each patientwas 1.2× their usual daily dose of basal Humulin NPH insulin tocompensate for the amount of short-acting insulin that they would notreceive on the test days. Blood samples were taken according to a setschedule over 13 hours. HDV was added to Humulin NPH insulin using themethod previously described to produce a suspension with a finalconcentration of 93.6 units of combined HDV Humulin R insulin and HDVHumulin NPH insulin/ml. The final suspension contained 0.52 mg of HDVlipid/ml. The patients were injected with the combined HDV-insulins onehour prior to the morning breakfast. At each of the three daily meals,breakfast, lunch and dinner, a 60 gram carbohydrate meal was prescribedby a dietitian.

The results of the experiments presented in this Experimental Exampleare now described. HDV-Humulin NPH insulin was well tolerated by thepatients and no adverse reactions were observed at the injection sites.Hypoglycemic reactions were not observed in patients receiving thistreatment. The blood glucose values of patients treated with HDV-HumulinNPH insulin are graphically presented in FIG. 21. FIG. 21 shows thatblood glucose concentrations increased, as anticipated, following mealsand glucose concentrations decreased over time until the next meal waseaten. This pattern was observed for all four patients. FIG. 22 showsthe effect of a single dose of HDV-Humulin NPH insulin on average bloodglucose concentrations in patients consuming three meals during the day.As with the individual patients, blood glucose concentrations increasedfollowing meals and glucose concentrations decreased over time until thenext meal was eaten. Average blood glucose concentrations were above thebaseline value at all time points. The curve suggests that the efficacyof HDV-Humulin NPH insulin improved throughout the day because there wasless variation between the high and low concentrations after the lunchand dinner meals than the breakfast meal. The effect of HDV-Humulin NPHinsulin on blood glucose concentrations over time relative to bloodglucose concentrations during fasting are shown in FIG. 23. Bloodglucose concentrations increased following meals then decreased overtime towards the glucose concentration during fasting until the nextmeal was eaten. Blood glucose concentrations were above fastingconcentrations throughout the study. Treatment of patients withHDV-Humulin NPH insulin resulted in some degree of post-prandial bloodglucose level control, indicating that HDV was able to carry sufficientquantities of Humulin NPH insulin to the liver at mealtimes to providethis control. Blood glucose levels were typical of Type I patients thatusually receive basal insulin therapy plus short-acting insulins at mealtimes.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations of theinvention may be devised by others skilled in the art without departingfrom the true spirit and scope of the invention. The appended claims areintended to be construed to include all such embodiments and equivalentvariations.

1-75. (canceled)
 76. A method of increasing the bioavailability of atleast one insulin in a patient, the method comprising: a. combining atleast one insulin with a lipid-based particle to form a constructcomprising the at least one insulin, wherein the lipid-based particle isdefined by a bipolar lipid membrane comprising lipids comprisingcholesterol, dicetyl phosphate, and an amphipathic lipid, and whereinthe bipolar lipid membrane further comprises a hepatocyte receptorbinding molecule, wherein the amphipathic lipid is at least one selectedfrom the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycero)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine; wherein the hepatocytereceptor binding molecule is the only hepatocyte receptor binder in thecomposition and is a biotin-containing compound selected from the groupconsisting of biotin DHPE (2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate), biotin-X-DHPE(2,3-diacetoxypropyl2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate), and any combinations thereof; wherein thebiotin-containing compound extends outward from the lipid-based particleand binds to a biotin-binding hepatocyte receptor; and, wherein the sizeof the lipid-based particle ranges from 0.0200 to 0.40 μm; and b.administering the construct containing the at least one insulin to thepatient.
 77. The method of claim 76, wherein the construct furthercomprises at least one charged organic molecule selected from the groupconsisting of protamines, polylysine, poly (arg-pro-thr)_(n), poly(DL-Ala-poly-L-lys)_(n), histones, sugar polymers comprising a primaryamino group, polynucleotides with primary amino groups, proteinscomprising amino acid residues with sulfhydral (S⁻) functional groups,and acidic polymers.
 78. The method of claim 76, wherein the at leastone insulin is at least one selected from the group consisting ofinsulin lispro, insulin aspart, regular insulin, insulin glargine,insulin zinc, human insulin zinc extended, isophane insulin, humanbuffered regular insulin, insulin glulisine, recombinant human regularinsulin, and recombinant human insulin isophane.
 79. The method of claim76, wherein the construct further comprises cellulose acetate phthalate.80. The method of claim 76, wherein the at least one insulin ischemically modified, thus having an isoelectric point that is distinctfrom the isoelectric point of the chemically unmodified insulin.
 81. Amethod of forming a time-release composition that provides increasedbiodistribution of insulin in a warm-bloodied subject, the methodcomprising: a. contacting (i) a bulk phase medium comprising aninsulin-containing construct and (ii) streptavidin agarose affinity-gel,in a system of pH equal to or greater than about 9.5, wherein theconstruct comprises insulin and a lipid-based particle defined by abipolar lipid membrane comprising lipids comprising cholesterol, dicetylphosphate, and an amphipathic lipid, wherein the amphipathic lipid is atleast one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycero)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine, wherein at least one of thelipids comprises iminobiotin or an iminobiotin derivative, wherein theat least one lipid comprising iminobiotin or an iminobiotin derivativeextends outward from the lipid-based particle, wherein the size of thelipid-based particle ranges from 0.0200 to 0.40 μm, whereby theinsulin-containing construct binds to the affinity-gel; b. separatingthe construct-bound affinity-gel from the bulk phase medium; and c.subjecting the construct-bound affinity-gel to a system of pH of around4.5, whereby the insulin-containing construct is released from theaffinity-gel, wherein the released construct contains insoluble insulin.82. The method of claim 81, wherein the construct further comprises atleast one charged organic molecule selected from the group consisting ofprotamines, polylysine, poly (arg-pro-thr)_(n), poly(DL-Ala-poly-L-lys)_(n), histones, sugar polymers comprising a primaryamino group, polynucleotides with primary amino groups, proteinscomprising amino acid residues with sulfhydral (S⁻) functional groups,and acidic polymers.
 83. The method of claim 81, wherein the at leastone insulin is at least one selected from the group consisting ofinsulin lispro, insulin aspart, regular insulin, insulin glargine,insulin zinc, human insulin zinc extended, isophane insulin, humanbuffered regular insulin, insulin glulisine, recombinant human regularinsulin, and recombinant human insulin isophane.
 84. The method of claim81, wherein the construct further comprises cellulose acetate phthalate.85. The method of claim 81, wherein the at least one insulin ischemically modified, thus having an isoelectric point that is distinctfrom the isoelectric point of the chemically unmodified insulin.
 86. Themethod of claim 81, wherein administration of the released construct tothe warm-bodied subject results in redissolution of the at least oneinsulin within the construct.